Lysosomal storage disease enzyme

Related application This application applies to U.S. Patent Provisional Application No. 61 / 343,177 filed on April 23, 2010, U.S. Patent Provisional Application No. 61 / 396,376 filed on May 26, 2010, September 9, 2010. US Patent Provisional Application No. 61 / 403,011 filed, US Patent Provisional Application No. 61 / 456,014 filed on October 29, 2010, US Patent Provisional Application No. 61 / filed on January 13, 2011 Claim the interests of No. 432,372. All the teachings of the above applications are incorporated herein by reference.

Lysosomal acid lipase (LAL) deficiency is an extremely rare lysosomal storage disease (LSD) characterized by deficient degradation of cholesteryl ester (CE) and triglyceride (TAG) within lysosomes due to enzyme deficiency. LAL deficiency is similar to other lysosomal storage disorders in which substrates accumulate in numerous tissues and cell types. In LAL deficiency, the accumulation of substrates in the cells of the reticular endothelial system, including Kupffer cells in the liver, histiocytes in the spleen and the lamina propria of the small intestine, is most pronounced. Reticuloendothelial cells express the macrophage mannose / N-acetylglucosamine receptor (the macrophage mannose receptor or MMR, also known as CD206), which binds to GlcNAc or a protein with mannose-terminal N-glycans. It mediates intracellular uptake and translocation into lysosomes, providing a pathway for potential correction of enzyme deficiency in the key cell types described above.

LAL deficiency is a polyphyletic disease with high morbidity and mortality, most often associated with gastrointestinal, hepatic and cardiovascular complications. The clinical effects of LAL deficiency are due to the accumulation of large amounts of lipid substances in lysosomes in many tissues and the severe disruption of cholesterol and lipid homeostasis mechanisms, including a significant increase in hepatic cholesterol synthesis. is there. There are at least two phenotypes of LAL deficiency: Wolman's disease (WD) and cholesteryl ester accumulation (CESD).

Wolman's disease is the most malignant symptom of LAL deficiency. This phenotype is characterized by onset in the gastrointestinal tract and liver, including stunted growth, malabsorption, steatorrhea, severe weight loss and hepatomegaly. Wolman's disease is rapidly progressive and is usually fatal within the first year of life. Case report reviews show that patients with growth deficiency due to LAL deficiency within the first year of life rarely survive longer than 12 months of age. In this most malignant form, growth failure is the most prominent clinical feature and a key factor in early mortality. Liver complications manifested by hepatomegaly and elevated transaminase are also common in infants. Physical findings include abdominal swelling due to hepatomegaly and splenomegaly, and radiological examination often reveals calcification of the adrenal glands. Laboratory evaluations usually reveal elevated blood transaminase levels and a deficiency or marked decrease in LAL enzyme activity. Elevated blood levels of cholesterol and triglycerides are also seen in patients.

Current treatment options for Wolman's disease are extremely limited. If there is evidence of fever and / or infection, administer antibiotics to the baby. Steroid replacement therapy and special nutritional supplements may be prescribed for adrenal insufficiency, but there is no evidence that these interventions prevent death, and it is currently unclear whether these will affect short-term survival. is there. In a series of four patients with LAL deficiency who were treated for bone marrow transplantation, all four died within months of the transplant due to treatment complications.

Patients with LAL deficiency may also develop significant liver and cardiovascular complications later in life, often referred to as cholesteryl ester accumulation disease (CESD). In CESD, the liver is severely affected by marked hepatomegaly, hepatocyte necrosis, elevated transaminase, cirrhosis and fibrosis. In LAL deficiency, hyperlipidemia and progressive atherosclerosis are also seen due to elevated CE and TG levels. Specifically, it is described that fatty deposits accumulate on the arterial wall shortly after birth. This deposit can narrow the arterial lumen and cause vascular occlusion, increasing the risk of serious cardiovascular events, including myocardial infarction and stroke. Symptoms of CESD are extremely diverse, and some patients may not be diagnosed until late adulthood complications, or may develop liver damage in early childhood. CESD is a serious health disorder associated with short life, and life expectancy in patients with CESD depends on the severity of complications.

Current treatment options for the CESD phenotype focus on controlling lipid accumulation in a diet that excludes cholesterol- and triglyceride-rich foods, and in suppressing cholesterol synthesis and apolipoprotein B production by administration of cholesterol-lowering drugs. .. Although clinical improvement may be seen, the onset of the underlying disease persists and the disease continues to progress.

Treatments for LAL deficiency often require lifelong treatment. In addition, due to the high cost of protein therapy, it is desirable to administer the least effective amount of therapeutic agent to treat LAL deficiency. However, at present, there is no effective treatment for patients with LAL deficiency, especially Wolman's disease and CESD. Therefore, there is a great need for effective treatments that minimize the frequency of administration in order to improve the quality of the patient's life. There is also a need for a highly expressed and robust protein production platform that is stable and capable of producing LAL proteins that are stable and efficiently targeted to the lysosomal compartments within the patient's affected histiocytes.

<p num="0009"> Disclosed herein are compositions of LAL that are particularly suitable for use in therapeutic methods, for example treating conditions associated with LAL deficiency. The LAL molecules described herein contain specific glycan structures that result in efficient and rapid uptake of cells into lysosomes when administered to a subject, eg, a human subject.</p><p num="0010"> In one aspect, the compositions disclosed herein comprise human LALs, and a significant proportion of human LALs undergo internal translocation by mannose-6-phosphate receptors on the cell surface, eg, on hepatocytes. Contains at least one mannose-6-phosphate glycan moiety that can act as a ligand for. In one embodiment, at least 30% of the LAL contained in the composition, eg, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. , At least 97% or at least 99% contain at least one mannose-6-phosphate moiety. The mannose-6-phosphate moiety is, for example, Asn of SEQ ID NO: 2.¹⁵, Asn⁵¹, Asn⁸⁰, Asn¹⁴⁰, Asn²⁵²And Asn³⁰⁰It can be seen on the N-glycan structure located at one or more residues selected from the group consisting of.</p><p num="0011"> In another aspect, the compositions disclosed herein contain human LALs, and a significant proportion of human LALs can interfere with the intracellular translocation of the enzyme into any of its N-glycan structures. Does not contain sialic acid moiety. In one embodiment, 15% or less of the LAL in the composition, such as 10% or less, 5% or less, 2% or less, 1% or less, contains or substantially has a sialic acid moiety in its N-glycan structure. All LALs do not contain sialic acid moieties.</p><p num="0012"> In another aspect, the compositions disclosed herein contain human LALs, and a significant proportion of human LALs do not contain fucose moieties in any of their N-glycan structures. In one embodiment, 50% or less of the LAL contained in the composition, for example, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 2% or less, 1% or less. Contains a fucose moiety in its N-glycan structure, or virtually all LALs do not contain a fucose moiety.</p><p num="0013"> In yet another aspect, vectors, host cells, expression systems and related methods suitable for producing LAL-containing compositions are described.</p><p num="0014"> In general, the LALs of the invention described and disclosed herein are human LALs. In one embodiment, the composition comprising LAL comprises mature LAL having the following amino acid sequence: SGGKLTAVDPETNMNVSEIISYWGFPSEEYLVETEDGYILCLNRIPHGRKNHSDKGPKPWFLQHGLLADSSNWVTNLANSSLGFILADAGFDVWMGNSRGNTWSRKHKTLSVSQDEFWAFSYDEMAKYDLPASINFILNKTGQEQVYYVGHSQGTTIGFIAFSQIPELAKRIKMFFALGPVASVAFCTSPMAKLGRLPDHLIKDLFGDKEFLPQSAFLKWLGTHVCTHVILKELCGNLCFLLCGFNERNLNMSRVDVYTTHSPAGTSVQNMLHWSQAVKFQKFQAFDWGSSAKNYFHYNQSYPPTYNVKDMLVPTAVWSGGHDWLADVYDVNILLTQINLVFHESIPEWEHLDFIWGLDAPWRLYNKIINLMRKYQ (SEQ ID NO: 2).</p><p num="0015"> In another embodiment, the mature LAL has the following amino acid sequence: GKLTAVDPETNMNVSEIISYWGFPSEEYLVETEDGYILCLNRIPHGRKNHSDKGPKPWFLQHGLLADSSNWVTNLANSSLGFILADAGFDVWMGNSRGNTWSRKHKTLSVSQDEFWAFSYDEMAKYDLPASINFILNKTGQEQVYYVGHSQGTTIGFIAFSQIPELAKRIKMFFALGPVASVAFCTSPMAKLGRLPDHLIKDLFGDKEFLPQSAFLKWLGTHVCTHVILKELCGNLCFLLCGFNERNLNMSRVDVYTTHSPAGTSVQNMLHWSQAVKFQKFQAFDWGSSAKNYFHYNQSYPPTYNVKDMLVPTAVWSGGHDWLADVYDVNILLTQINLVFHESIPEWEHLDFIWGLDAPWRLYNKIINLMRKYQ (SEQ ID NO: 3).</p><p num="0016"> In another embodiment, the mature LAL has the following amino acid sequence: TAVDPETNMNVSEIISYWGFPSEEYLVETEDGYILCLNRIPHGRKNHSDKGPKPWFLQHGLLADSSNWVTNLANSSLGFILADAGFDVWMGNSRGNTWSRKHKTLSVSQDEFWAFSYDEMAKYDLPASINFILNKTGQEQVYYVGHSQGTTIGFIAFSQIPELAKRIKMFFALGPVASVAFCTSPMAKLGRLPDHLIKDLFGDKEFLPQSAFLKWLGTHVCTHVILKELCGNLCFLLCGFNERNLNMSRVDVYTTHSPAGTSVQNMLHWSQAVKFQKFQAFDWGSSAKNYFHYNQSYPPTYNVKDMLVPTAVWSGGHDWLADVYDVNILLTQITNLVFHESIPEWEHLDFIWGLDAPWRLYNKIINLMRKYQ (SEQ ID NO: 4).</p><p num="0017"> In another embodiment, the mature LAL has the following amino acid sequence: AVDPETNMNVSEIISYWGFPSEEYLVETEDGYILCLNRIPHGRKNHSDKGPKPWFLQHGLLADSSNWVTNLANSSLGFILADAGFDVWMGNSRGNTWSRKHKTLSVSQDEFWAFSYDEMAKYDLPASINFILNKTGQEQVYYVGHSQGTTIGFIAFSQIPELAKRIKMFFALGPVASVAFCTSPMAKLGRLPDHLIKDLFGDKEFLPQSAFLKWLGTHVCTHVILKELCGNLCFLLCGFNERNLNMSRVDVYTTHSPAGTSVQNMLHWSQAVKFQKFQAFDWGSSAKNYFHYNQSYPPTYNVKDMLVPTAVWSGGHDWLADVYDVNILLTQITNLVFHESIPEWEHLDFIWGLDAPWRLYNKIINLMRKYQ (SEQ ID NO: 19).</p><p num="0018"> In another embodiment, the mature LAL is a mixture of at least two polypeptides selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 19.</p><p num="0019"> The present invention also provides a composition containing an isolated mixture of useful protein molecules of individual types, such as the proteins disclosed herein, one or more contained in the mixture. A specific oligosaccharide structure, specifically the oligosaccharide structure disclosed herein, is attached to the protein molecule. For example, the present invention provides an isolated mixture of LAL molecules, eg, human LAL molecules, containing LAL molecules glycosylated by one or more of the following structures An ~ On:<chemistry num="1"><img id="000003" he="70" wi="161" file="JP2016190867A_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>Square = N-acetylglucosamine Black square = mannose-6-phosphate = Mannose Black circle = galactose Black triangle = fucose.</p><p num="0020"> In one aspect of the invention, the composition comprises any isolated individual polypeptide or combination of the polypeptides. In one embodiment, the composition is such that the pharmaceutical composition, eg, the composition, is suitable for administration, eg, to a subject (eg, a human, particularly a patient suffering from or diagnosed with a condition). , Can be a formulation further comprising a pharmaceutically acceptable carrier. The composition can be administered in a variety of ways, including intravenous administration. In another embodiment, the composition can further comprise a second agent. Such agents can be drugs, or agents that can influence or regulate biological processes when administered to a subject. For example, the second agent can be an immunomodulator. Such immunomodulators, when administered with any of the LAL compositions described herein (ie, simultaneously, or immediately before or after), are immunogens of the LAL composition in the subject. Any drug that may have a sex-reducing effect (eg, rituximab, or any other B cell depleting antibody) can be mentioned.</p><p num="0021"> In the final aspect, methods and compositions for treating symptoms associated with LAL deficiency are disclosed.</p><p num="0022"> Further reconsideration of the following detailed description in conjunction with the accompanying drawings and arrangement will clarify further objects and aspects of the invention. For example, the present invention provides the following items. (Item 1) A composition comprising an isolated human recombinant lysosomal acid lipase (LAL) comprising one or more N-glycan structures. (Item 2) The composition according to item 1, wherein the LAL substantially comprises the amino acid sequence of SEQ ID NO: 2. (Item 3) The composition according to item 2, wherein the LAL comprises the amino acid sequence set forth in SEQ ID NO: 2. (Item 4) The LAL is Asn of SEQ ID NO: 2.¹⁵, Asn⁵¹, Asn⁸⁰, Asn¹⁴⁰, Asn²⁵²And Asn³⁰⁰The composition according to item 3, which is N-linked glycosylated at at least one position selected from the group consisting of. (Item 5) The LAL is Asn of SEQ ID NO: 2.¹⁵, Asn⁸⁰, Asn¹⁴⁰, Asn²⁵²And Asn³⁰⁰The composition according to item 4, which is N-glycosylated in. (Item 6) The LAL has an N-glycan structure containing phosphorylated mannose as Asn of SEQ ID NO: 2.⁸⁰, Asn¹⁴⁰Or Asn²⁵²The composition according to item 5, which is contained in. (Item 7) The LAL has an N-glycan structure containing phosphorylated mannose.⁸⁰The composition according to item 5, which is contained in. (Item 8) Asn⁸⁰5. The composition according to item 5, wherein at least 30% of the N-glycan structure associated with has phosphorylated mannose. (Item 9) Asn⁸⁰5. The composition according to item 5, wherein at least 50% of the N-glycan structure associated with the phosphorylated mannose. (Item 10) The LAL has an N-glycan structure containing mannose diphosphate.⁸⁰The composition according to item 5, which is contained in. (Item 11) Asn⁸⁰The composition according to item 5, wherein at least 50% of the N-glycan structure associated with contains mannose diphosphate. (Item 12) The LAL has an N-glycan structure containing phosphorylated mannose (M6P).¹⁴⁰The composition according to item 5, which is contained in. (Item 13) Asn¹⁴⁰The composition according to item 5, wherein about 10% to about 50% of the N-glycan structure associated with contains M6P. (Item 14) The LAL has an N-glycan structure containing phosphorylated mannose (M6P).²⁵²The composition according to item 5, which is contained in. (Item 15) Asn²⁵²The composition according to item 14, wherein at least 50% of the N-glycan structure in the above contains M6P. (Item 16) The LAL has an N-glycan structure containing a high mannose group.⁸⁰Or Asn²⁵²The composition according to item 5, which is contained in. (Item 17) The composition according to item 16, wherein the high mannose group comprises 6, 7, 8, 9 or 10 mannoses. (Item 18) 17. The composition of item 17, wherein the high mannose group comprises 7, 8 or 9 mannose. (Item 19) The LAL Asn an N-glycan structure containing 7, 8 or 9 mannoses.⁸⁰The composition according to item 18, which is included in. (Item 20) Asn⁸⁰The composition according to item 18, wherein at least 80% of the N-glycan structure in is comprising 7, 8 or 9 mannoses. (Item 21) The LAL Asn an N-glycan structure containing 7, 8 or 9 mannoses.²⁵²The composition according to item 18, which is included in. (Item 22) Asn²⁵²The composition according to item 18, wherein at least 70% of the N-glycan structure in is comprising 7, 8 or 9 mannoses. (Item 23) The LAL has an N-glycan structure containing terminal galactose.¹⁵, Asn¹⁴⁰Or Asn³⁰⁰The composition according to item 5, which is contained in. (Item 24) Asn¹⁵The composition according to item 5, wherein about 2% to about 10% of the N-glycan structure associated with contains terminal galactose. (Item 25) Asn¹⁴⁰The composition according to item 5, wherein less than 5% of the N-glycan structure associated with contains terminal galactose. (Item 26) Asn³⁰⁰The composition according to item 5, wherein less than 100% of the N-glycan structure associated with contains terminal galactose. (Item 27) Asn of the LAL⁵¹5. The composition according to item 5, wherein is non-glycosylated or substantially non-glycosylated. (Item 28) The composition according to item 1, wherein the LAL contains an N-glycan structure without xylose, and less than 15% of the N-glycan structure contains sialic acid. (Item 29) The composition according to item 1, wherein the LAL contains an N-glycan structure without xylose, and less than 10% of the N-glycan structure contains sialic acid. (Item 30) The composition according to item 1, wherein the LAL contains an N-glycan structure without xylose, and less than 5% of the N-glycan structure contains sialic acid. (Item 31) The composition according to item 1, wherein the LAL contains an N-glycan structure without xylose, and less than 1% of the N-glycan structure contains sialic acid. (Item 32) The composition according to item 1, wherein the LAL is substantially free of sialic acid and xylose. (Item 33) Item 1 in which the LAL contains an N-glycan structure, and less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or less than 1% of the N-glycan structure contains fucose. The composition according to. (Item 34) The composition according to item 1, wherein the LAL does not contain fucose. (Item 35) Item 1 wherein the LAL contains an N-glycan structure and at least 30%, 50%, 60%, 70%, 80%, 90% and 95% of the N-glycan structure contains phosphorylated mannose (M6P). The composition described. (Item 36) The composition according to item 1, wherein the LAL contains an N-glycan structure, and at least 90% of the N-glycan structure contains phosphorylated mannose (M6P). (Item 37) The composition according to item 5, wherein the LAL comprises the following N-linked glycosylation profile: g) Asn¹⁵In GlcNAc4Man3 GlcNAc2 or Gal1GlcNAc4Man3GlcNAc2; h) Asn⁸⁰In Phos2Man7GlcNAc2; i)¹⁴⁰In Phos1Man6GlcNAc2, GlcNAc1Phos1Man6GlcNAc2, Man3GlcNAc2, GlcNAc2Man3GlcNAc2, GlcNAc3Man3GlcNAc2, GlcNAc4Man3 GlcNAc2 or Gal1GlcNAc4Man3GlcNAc2; j) Asn²⁵²In Man7GlcNAc2, Man8GlcNAc2, Man9GlcNAc2, Phos1Man8GlcNAc2 or Phos1Man9GlcNAc2; and k) Asn³⁰⁰In GlcNAc2Man3GlcNAc2, GlcNAc3Man3GlcNAc2, GlcNAc4Man3GlcNAc2, Gal1GlcNAc4Man3GlcNAc2, GlcNAc5Man3GlcNAc2, Gal1GlcNAc5Man3GlcNAc2, GlcNAc6Man3 GlcNAc2 or Gal1GlcNAc6Man3GlcNAc2 (During the ceremony, Man = Mannose, GlcNAc = N-acetylglucosamine, Phos = Phosphoric Acid, Gal = galactose Is). (Item 38) The LAL is Asn of SEQ ID NO: 2.¹⁵, Asn⁵¹, Asn⁸⁰, Asn¹⁴⁰, Asn²⁵²And Asn³⁰⁰The composition according to item 1, which is N-linked glycosylated in. (Item 39) The composition according to item 1, wherein the LAL comprises an N-glycan selected from the following structures: (Chemical 1)<img id="000004" he="60" wi="160" file="JP2016190867A_D0001.tif" img-format="tif" img-content="drawing" />(During the ceremony, Square = N-acetylglucosamine Black square = mannose-6-phosphate Maru = Mannose Black circle = galactose Black triangle = fucose Is). (Item 40) 39. The composition of item 39, wherein the LAL is produced in germline transgenic birds. (Item 41) 40. The composition of item 40, wherein the LAL is produced from the oviduct cells of the germline transgenic bird. (Item 42) The composition according to item 1, wherein the oligosaccharide structure is derived from a bird. (Item 43) 42. The composition of item 42, wherein the chicken is a chicken. (Item 44) A composition comprising a mixture of recombinant human lysosomal acid lipase (LAL), wherein the mixture comprises at least two human LALs selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 19. .. (Item 45) 44. The composition of item 44, wherein the mixture of human LAL comprises N-glycans selected from the following structures: (Chemical 2)<img id="000005" he="61" wi="160" file="JP2016190867A_D0001.tif" img-format="tif" img-content="drawing" />(During the ceremony, Square = N-acetylglucosamine Black square = mannose-6-phosphate Maru = Mannose Black circle = galactose Black triangle = fucose Is). (Item 46) 44. The composition according to item 44, wherein the LAL comprises the following N-linked glycosylation profile: a) Asn of SEQ ID NOs: 2, 3, 4 and 19, respectively¹⁵, Asn¹³, Asn¹⁰And Asn⁹In GlcNAc4Man3 GlcNAc2 or Gal1GlcNAc4Man3GlcNAc2; b) Asn of SEQ ID NOs: 2, 3, 4 and 19, respectively⁸⁰, Asn⁷⁸, Asn⁷⁵And Asn⁷⁴In Phos2Man7GlcNAc2; c) Asn of SEQ ID NOs: 2, 3, 4 and 19, respectively¹⁴⁰, Asn¹³⁸, Asn¹³⁵And Asn¹³⁴In Phos1Man6GlcNAc2, GlcNAc1Phos1Man6GlcNAc2, Man3GlcNAc2, GlcNAc2Man3GlcNAc2 GlcNAc3Man3GlcNAc2 GlcNAc4Man3 GlcNAc2 or Gal1GlcNAc4Man3GlcNAc2; d) Asn of SEQ ID NOs: 2, 3, 4 and 19, respectively²⁵², Asn²⁵⁰, Asn²⁴⁷And Asn²⁴⁶In Man7GlcNAc2, Man8GlcNAc2, Man9GlcNAc2, Phos1Man8GlcNAc2 or Phos1Man9GlcNAc2; and e) Asn of SEQ ID NOs: 2, 3, 4 and 19, respectively³⁰⁰, Asn²⁹⁸, Asn²⁹⁵And Asn²⁹⁴In GlcNAc2Man3GlcNAc2, GlcNAc3Man3GlcNAc2, GlcNAc4Man3GlcNAc2, Gal1GlcNAc4Man3GlcNAc2, GlcNAc5Man3GlcNAc2, Gal1GlcNAc5Man3GlcNAc2, GlcNAc6Man3 GlcNAc2 or Gal1GlcNAc6Man3GlcNAc2. (Item 47) A composition comprising an isolated lysosomal acid lipase (LAL) molecule, wherein the LAL is produced in the oviduct cells of the transgenic chicken and is isolated from the egg white of the transgenic chicken containing the transgene encoding the LAL. (Item 48) 47. The composition of item 47, wherein the LAL is a human LAL. (Item 49) The composition according to item 48, wherein the oviduct cells are tubular gland cells. (Item 50) 49. The composition of item 49, wherein the isolated human LAL comprises a chicken-derived glycosylation pattern. (Item 51) A method of treating a patient suffering from a condition associated with LAL deficiency, which comprises administering to the patient a therapeutically effective amount of a composition comprising recombinant human LAL. (Item 52) 51. The method of item 51, wherein the recombinant human LAL is the composition according to any one of items 1-33. (Item 53) 51. The method of item 51, wherein the condition is cholesteryl ester accumulation disease (CESD). (Item 54) 51. The method of item 51, wherein the condition is Wolman's disease. (Item 55) 51. The method of item 51, wherein the patient is administered the LAL composition between about 0.35 mg / kg and about 5.0 mg / kg. (Item 56) 51. The method of item 51, wherein the patient is administered the LAL composition between about 0.35 mg / kg and about 3.0 mg / kg. (Item 57) 51. The method of item 51, wherein the patient is administered once every 7 days to once every 45 days. (Item 58) For the patients, once every 3 days, once every 5 days, once a week, once every 2 weeks, once every 20 days, once every 28 days, once every 30 days or every month 51. The method of item 51, which is administered once. (Item 59) 51. The method of item 51, wherein an effective amount between about 1.0 mg / kg and 3.0 mg / kg is administered to the patient every other week. (Item 60) A method for producing glycosylated human lysosomal acid lipase (LAL), which is a germline transgenic that contains a transgene encoding a human LAL operably linked to a promoter and expresses the human LAL in an egg transfusion tube cell. A method of producing a bird, wherein the LAL is glycosylated in the transgene tube cell and accumulated in the hard shell egg laid by the transgenic bird. (Item 61) A transgenic bird that produces glycosylated human lysosomal acid lipase (LAL). (Item 62) Eggs laid by the transgenic birds according to item 61. (Item 63) Egg white containing human LAL produced by the transgenic bird according to item 61. (Item 64) A vector carrying a nucleic acid sequence encoding the human recombinant lysosomal acid lipase according to item 1. (Item 65) Host cells transformed with the vector according to item 64. (Item 66) A method of treating a patient suffering from a condition associated with LAL deficiency, in which a therapeutically effective amount of a composition comprising recombinant human LAL is administered to the patient and a second therapeutic agent is administered to the patient. Methods, including administration to. (Item 67) 66. The method of item 66, wherein the second therapeutic agent is a cholesterol lowering agent selected from the group consisting of atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin. (Item 68) 66. The method of item 66, wherein the second therapeutic agent is an immunosuppressant. (Item 69) A pharmaceutical preparation comprising the composition according to item 1 together with a pharmaceutically acceptable carrier, diluent or additive. (Item 70) 29. The pharmaceutical formulation of item 69, comprising at least one agent selected from the group consisting of trisodium citrate dehydration, citric acid and human serum albumin. (Item 71) The pharmaceutical preparation according to item 69, which is provided in an aqueous solution whose pH is maintained between about 5.6 and about 6.2. (Item 72) The pharmaceutical preparation according to item 71, wherein the pH is maintained between 5.7 and 6.1.</p>

<figref num="1">It is a figure which shows the amino acid sequence of human LAL. The amino acid sequence of recombinant hLAL shows 100% homology with the sequence of native human LAL. The mature hLAL is underlined.</figref><figref num="2">It is a figure which shows the nucleotide sequence of the recombinant hLAL which is the rhLAL transgene of pALVIN-OVR1-I-hLAL-dSA.</figref><figref num="3">Figures 3A and 3B. It is a schematic diagram of pALVIN-OVR1-I-hLAL-dSA and its provirus region. FIG. 3A is a schematic representation of the human LAL retrovirus expression vector used to generate transduced particles (plasmid DNA sequence in Attachment A). FIG. 3A is a diagram of the provirus region of pALVIN-OVR1-I-hLAL-dSA integrated into the genome. SIN LTR is a self-activated long terminal repeat; OV DHSIII enhancer is a DNase hypersensitive site of the ovoalbumin gene III; OV intron is an ovoalbumin 5'untranslated region and intron 1; hLAL is a human LAL cDNA; OV3' UTR is the ovoalbumin gene 3'untranslated region; partial gag is the partial gag gene; LTR is the long terminal repeat sequence.</figref><figref num="4-1">Figure 4. It is a figure which shows the nucleotide sequence of pALVIN-OVR1-I-hLAL-dSA.</figref><figref num="4-2">Figure 4. It is a figure which shows the nucleotide sequence of pALVIN-OVR1-I-hLAL-dSA.</figref><figref num="4-3">Figure 4. It is a figure which shows the nucleotide sequence of pALVIN-OVR1-I-hLAL-dSA.</figref><figref num="4-4">Figure 4. It is a figure which shows the nucleotide sequence of pALVIN-OVR1-I-hLAL-dSA.</figref><figref num="5-1">Figure 5. It is a figure which shows the nucleotide sequence of the pALVIN-OVR1-I-hLAL-dSA provirus region integrated into the genome.</figref><figref num="5-2">Figure 5. It is a figure which shows the nucleotide sequence of the pALVIN-OVR1-I-hLAL-dSA provirus region integrated into the genome.</figref><figref num="5-3">Figure 5. It is a figure which shows the nucleotide sequence of the pALVIN-OVR1-I-hLAL-dSA provirus region integrated into the genome.</figref><figref num="6-1">Figure 6. It is a figure which shows the nucleotide sequence of pALVIN-OV-1.1-I vector.</figref><figref num="6-2">Figure 6. It is a figure which shows the nucleotide sequence of pALVIN-OV-1.1-I vector.</figref><figref num="6-3">Figure 6. It is a figure which shows the nucleotide sequence of pALVIN-OV-1.1-I vector.</figref><figref num="6-4">Figure 6. It is a figure which shows the nucleotide sequence of pALVIN-OV-1.1-I vector.</figref><figref num="7">It is a figure which shows the nucleotide sequence of rhLAL adapter.</figref><figref num="8">It is a figure which shows the nucleotide sequence of rhLAL containing a partial ovalbumin promoter.</figref><figref num="9">It is a figure which shows the nucleotide sequence of the OVR1 promoter.</figref><figref num="10">It is a schematic diagram of the stage used to construct the pALVIN-OVR1-I-hLAL-dSA vector.</figref><figref num="11">It is a figure which shows the real-time PCR analysis of the blood DNA sample derived from the hemizygous transgenic G1 progeny of XLL109. The signal from the replicated DNA sample of 1LL7466, a hemizygous G1 progeny, is indicated by a curve in which the increase in delta Rn begins before cycle 22. Curves for two non-transgenic offspring are shown, but these curves remain near baseline for at least 34 cycles.</figref><figref num="12">Figures 12A-12D. It is a figure which shows the Southern analysis of the G1 chicken carrying the ALVIN-OVR1-I-hLAL-dSA transgene. FIG. 12A shows a schematic representation of the integrated transgene, with adjacent genomic regions shown along with known locations of the transgene BlpI site and predicted locations of the adjacent genomic BlpI site. The positions of the OV promoter probe and hLAL coding sequence probe (hLAL probe) are indicated by black lines. The 4.3 kb and 10.6 kb positions detected by Southern analysis are shown along with the predicted size of the 4.3 kb and 10.6 kb band genomic and transgene moieties. FIG. 12B illustrates a Southern blot of genomic DNA digested with BlpI and probed with an OV probe. WT CTRL is genomic DNA isolated from non-transgenic chickens. Above the lane is the G1 transgenic ID number. The position and size (kb) of the molecular weight marker is shown on the left side of the blot. The location and size of the detected transgene fragment (4.3 kb) and endogenous ovalbumin gene (4.1 kb) are shown on the right side of the blot. FIG. 12C is a diagram showing a Southern blot probed with an hLAL probe. The location and size of the detected transgene fragment (10.6 kb) is shown on the right side of the blot. FIG. 12D is a magnified view of a portion of the figure shown in FIG. 12B to show the presence of the 4.1 kb and 4.3 kb bands.</figref><figref num="13">Figure 13A. It is a schematic diagram of the ALVIN-OVR1-I-hLAL-dSA transgene. It also shows the size of the ApaLI band that is expected to be detected by the OV and hLAL probes. FIG. 13B is a schematic diagram of Southern blot analysis of the ALVIN-OVR1-I-hLAL-dSA transgene for confirmation of transgene size. Southern blots of genomic DNA were digested with ApaLI and probed with an OV probe (left panel) or hLAL probe (right panel). WT CTRL is genomic DNA isolated from non-transgenic chickens. The G1 ID number is shown above each lane. The position and size (kb) of the molecular weight marker is shown on the left side of the blot. On the right side of the blot, the location and size of the detected transgene fragment (OV promoter probe, 3.6 kb; hLAL probe, 3.8 kb) and the endogenous ovalbumin gene (7.7 kb) are shown.</figref><figref num="14">It is a figure which shows the strain of a transgenic chicken. The number of generations (G0, G1 or G2), identification number, gender and hatching date of each chicken are shown. Other G1 chickens are of other strains.</figref><figref num="15">It is a figure which shows the purification step of hLAL from egg white.</figref><figref num="16">It is a figure which represents N-glycan which is seen as an N-linked glycosylation structure in LAL produced according to this invention. Squares are N-acetylglucosamine; black squares are mannose-6-phosphate; circles are mannose; black circles are galactose; black triangles are fucose.</figref><figref num="17">It is a figure which showed the relative position of the predicted N-glycan site on the LAL polypeptide (arrow) described by SEQ ID NO: 1. The typical structure of N-glycans detected at each site is shown. Squares are N-acetylglucosamine; black squares are mannose-6-phosphate; circles are mannose; black circles are galactose; black triangles are fucose.</figref><figref num="18">It is a figure showing the phosphorylated N-glycan dissociated by PNGase and analyzed by MALDI-TOF. The structure is shown.</figref><figref num="19">It is a figure which shows the effect of dephosphorylation of LAL on HPAEC-PAD retention time of N-glycan. LALs produced according to the present invention were either dephosphorlated with bacterial alkaline phosphatase (upper panel) or left untreated (lower panel). The dissociated N-glycans were analyzed by HPAEC-PAD.</figref><figref num="20">It is a figure showing the co-localization of the recombinant human LAL (SBC-102) and the lysosomal marker in the lysosome of the cell examined by the confocal fluorescence microscope using the continuous scan mode.</figref><figref num="21">FIG. 5 shows the binding specificity of recombinant human LAL (SBC-102) to the GlcNAc / mannose receptor evaluated by a competitive binding assay using the macrophage cell line NR8383.</figref><figref num="22">It is a figure which shows the activity of recombinant human LAL in a cell in normal and LAL deficient cells in vitro.</figref><figref num="23">It is a figure which shows the effect of the recombinant human LAL (SBC-102) treatment on the internal organ mass of a LAL-deficient rat. Organ size is LAL^-/-Rat and LAL^+/+In rats, solvent or SBC-102 is administered at 5 mg / kg once weekly for 4 weeks and then expressed as a percentage of body weight determined at 8 weeks of age.</figref><figref num="24">Solvent or SBC-102 5 mg / kg^-1It is a figure showing the body weight of the wild type and LAL-deficient rats after administration once a week for 4 weeks. On the X-axis, the administration is highlighted by a diamond starting at week 4.</figref><figref num="25">Solvent or recombinant human LAL (SBC-102) 5 mg · kg in WT and LAL-deficient rats^-1It is a figure which shows the cholesterol, cholesteryl ester and triglyceride level of the liver determined at the age of 8 weeks after administration once a week for 4 weeks.</figref><figref num="26">FIG. 5 represents the percentage of weight gain determined at 8 weeks of age after 4 weeks of administration of recombinant human LAL (SBC-102) at the indicated levels and schedule in LAL-deficient rats.</figref><figref num="27">FIG. 5 shows liver weight expressed as a percentage of body weight determined at 8 weeks of age after administration of SBC-102 at the indicated levels and schedule for 4 weeks in LAL-deficient rats.</figref><figref num="28">FIG. 5 shows tissue cholesteryl ester levels determined at 8 weeks of age after 4 weeks of administration of SBC-102 at the indicated levels and schedule.</figref><figref num="29">It is a figure which shows the progress of the daily weight gain of the rat which received 1 mg / kg LAL per week, 5 mg / kg LAL per week or 5 mg / kg LAL per 2 weeks.</figref><figref num="30">It is a diagram showing a gross pathological examination of a treated individual, showing that the size and color of the liver have become considerably normal, as seen in the dissection of the upper panel. The lower panel is also a histopathological examination of liver tissue, where the LAL liver tissue of treated rats shows normal liver tissue, in stark contrast to placebo-treated rats, which have a significant accumulation of foamy macrophages. is there.</figref>

Definition Certain definitions are provided herein to explain and define the meaning and scope of the various terms used herein to describe the invention.

The term "acceptable" with respect to a formulation, composition or ingredient as used herein has a lasting adverse effect on the general health of the subject being treated, as used herein. It means not having it.

As used herein, the term "administration" refers to giving the recombinant human lysosomal acid lipase of the invention to a subject in need of treatment.

The "nucleic acid" or "polynucleotide sequence" includes, but is not limited to, eukaryotic mRNA, cDNA, genomic DNA and synthetic DNA and RNA sequences including the natural nucleoside bases adenine, guanine, cytosine, thymidine and uracil. To do. The term also includes sequences with one or more modified bases.

As used herein, the term "bird" refers to any species, subspecies or variety of taxonomic ava species, such as chickens, cassowaries, ducks, geese, quails, quails, etc. Refers to parrots, finch, hawks, quails, and running birds including ostriches, emu and cassowaries. The term chicken (Gal1us) Gal1us) (eg White Leghorn, Brown Leghorn, Barred-Rock, Sussex, New Hampshire, Rhode Island, Australorp, Minorca, Amrocks) Includes various known strains of (Amrox), California Gray, and other poultry commonly bred in commercial quantities, including citrus, pheasant, quail, duck, ostrich, and embryos and fetuses. Individual avian organisms at all developmental stages, including the stage of, are also included in this term.

A "therapeutic protein" or "pharmaceutical protein" includes an amino acid sequence that occupies all or part of a drug.

A "coding sequence" or "open reading frame" can be transcribed in vitro or in vivo and translated into a polypeptide (in the case of DNA) or poly when placed under the control of a suitable regulatory sequence. Refers to a polynucleotide sequence or nucleic acid sequence that can be translated into a peptide (in the case of mRNA). The coding sequence boundaries are defined by the 5'(amino) -terminal translation initiation codon and the 3'(carboxy) -terminal translation stop codon. The transcription termination sequence is usually located on the 3'side of the coding sequence. Untranslated regions may be adjacent to the 5'and / or 3'ends of the coding sequence.

"Exon" refers to the portion of a gene that is "expressed" in cytoplasmic mRNA after removal of an intron or intervening sequence by nuclear splicing when transcribed into a nuclear transcript.

Nucleic acid "regulatory sequences" or "regulatory sequences" are promoter sequences necessary and sufficient for transcription and translation of a given coding sequence in a defined host cell, translation initiation and stop codons, ribosome binding sites, polyadenylation signals, Refers to transcription termination sequences, upstream regulatory domains, enhancers, etc. Examples of control sequences suitable for eukaryotic cells are promoters, polyadenylation signals and enhancers. Not all of these regulatory sequences must be present in the recombinant vector, as long as the regulatory sequences necessary and sufficient for transcription and translation of the desired gene are present.

By "operably linked" is meant that the coding and control sequences are arranged to perform the desired function. Thus, a control sequence that is operably linked to a coding sequence can result in expression of the coding sequence. When DNA polymerase binds to a promoter sequence and transcribes the coding sequence into an mRNA that can be translated into a coding protein, the coding sequence is operably linked to or is under the control of a transcriptional regulatory region in the cell. It will be. As long as the control sequence functions to direct the expression of the coding sequence, the control sequence need not be adjacent to the coding sequence. Thus, for example, an untranslated but transcribed intervening sequence may be present between the promoter and coding sequences, yet the promoter sequence is considered to be "operably linked" to the coding sequence. Can be done.

The terms "heterologous" and "foreign" associated with nucleic acid sequences, such as coding and regulatory sequences, are usually not associated with regions of recombinant constructs or specific chromosomal loci, and / or with specific cells. Represents an array that is usually unrelated. Thus, the "foreign" region of a nucleic acid construct is a segment of identifiable nucleic acid that is not found together in nature and is within or bound to another nucleic acid molecule. For example, the exogenous region of a construct can include coding sequences that are flanked by sequences that are not naturally found with the coding sequences. Another example of an extrinsic coding sequence is a construct in which the coding sequence itself is not found naturally (eg, a synthetic sequence having a codon different from that of the native gene). Similarly, a host cell transformed with a construct or nucleic acid that is not normally present in the host cell is considered exogenous for the purposes of the present invention.

The terms "N-glycan", "oligosaccharide", "oligosaccharide structure", "glycosylation pattern", "glycosylation profile" and "glycosylation structure" as used herein are substantially synonymous. , Each refer to one or more structures formed from sugar residues and bound to glycosylated proteins.

As used herein, "foreign protein" refers to a protein that is not naturally present in a particular tissue or cell, a protein that is an expression product of an exogenous expression construct or transgene, or a particular tissue or cell. Refers to a protein that does not exist in nature in a given amount. Proteins that are exogenous to the egg are proteins that are not normally found in the egg. For example, a protein that is exogenous to an egg can be a protein that is present in the egg as a result of expression of a coding sequence that is present in the transgene of the egg-laying animal.

"Intrinsic gene" refers to a natural gene or fragment thereof that is normally associated with a particular cell.

"LAL" means "human lysosomal acid lipase," "SBC-102," or "human lysosomal acid lipase molecule," and these terms are used interchangeably throughout this specification.

The expression products described herein can be composed of a proteinaceous substance having a defined chemical structure. However, the detailed structure is determined by a number of factors, especially chemical modifications common to proteins. For example, all proteins contain an ionizable amino group and a carboxyl group, so that the protein can be in acidic or basic salt or neutral form. The primary amino acid sequence is another chemical derivative that results from the use of sugar molecules (glycosylation) or often by binding to sugars, including covalent or ionic bonds, such as lipids, phosphates, acetyl groups, etc. It can be derivatized by chemistry. These modifications can occur in vitro or in vivo, the latter being performed by the host cell through a post-translational processing system. Such modifications may increase or decrease the biological activity of the molecule, and such chemically modified molecules shall also be within the scope of the present invention.

Alternative methods of cloning, amplification, expression and purification will be apparent to those of skill in the art. Representative methods are disclosed in Sambrook, Fritsch and Maniatis, Molecular Cloning, a Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory (1989).

"Vector" means a polynucleotide composed of single-stranded, double-stranded, circular, or supercoiled DNA or RNA. A typical vector may consist of the following elements operably linked at appropriate distances to allow functional gene expression: origin of replication, promoter, enhancer, 5'mRNA leader sequence, ribosome binding site, Nucleic acid cassette, termination and polyadenylation sites, and selectable marker sequences. For certain applications, one or more of the above elements may not be present. The nucleic acid cassette may include a restriction site for inserting the expressed nucleic acid sequence. In a functional vector, the nucleic acid cassette comprises an expressed nucleic acid sequence having translation initiation and termination sites. Arbitrarily, the intron may be included in the structure, for example, on the 5'side of the coding sequence. The vector is arranged such that a particular coding sequence is placed in the vector with the appropriate regulatory sequence and the coding sequence is the position and orientation with which the coding sequence is transcribed relative to the control sequence or under "control" of the regulatory sequence. To construct. In order to obtain such results, it may be desirable to modify the sequence encoding a particular protein of interest. For example, in some cases it may be necessary to modify the sequence so that it can bind to the control sequence in the appropriate direction, or to maintain a reading frame. Control sequences and other regulatory sequences may be ligated with the coding sequence prior to insertion into the vector. Alternatively, the coding sequence may be cloned directly into an expression vector that already contains the control sequence and a suitable restriction site within the reading frame that has the control sequence and is under its regulatory control.

A "promoter" is a site on DNA where RNA polymerase binds and initiates transcription of a gene. In some embodiments, the promoter can be modified by the addition or deletion of sequences or replaced with another sequence that includes native and synthetic sequences as well as sequences that can be a combination of synthetic and native sequences. Many eukaryotic promoters contain two types of recognition sequences: the TATA box and the upstream promoter element. The former is located upstream of the transcription initiation site and is involved in directing RNA polymerase to initiate transcription at the correct site, while the latter is thought to determine the transcription rate and is upstream of the TATA box. Enhancer elements can also stimulate transcription from linked promoters, but many function only in certain cell types. Enhancer / promoter elements from many viruses, such as the SV40 promoter, cytomegalovirus (CMV) promoter, Laus sarcoma virus (RSV) promoter and murine leukemia virus (MLV) promoter, are all active in a wide range of cell types and are " It is called "unevenly distributed". Alternatively, non-constitutive promoters such as the mouse mammary tumor virus (MMTV) promoter can also be used in the present invention. The nucleic acid sequence inserted at the cloning site may have any open reading frame encoding the polypeptide of interest, but if the coding sequence encodes the polypeptide of interest, it may block the production of the appropriate mRNA molecule, And / or should lack potential splicing sites capable of producing abnormally spliced or abnormal mRNA.

As used herein, the term "pharmaceutical composition" refers to the compounds described herein and other chemical components such as carriers, stabilizers, diluents, dispersants, suspending agents, thickening agents. Refers to a mixture with an agent and / or an excipient.

The term "poultry-derived" or "bird-derived" refers to a composition or substance produced or obtained from poultry. Poultry refers to birds that can be bred as livestock, including, but not limited to, chickens, ducks, turkeys, quails and running birds. For example, "poultry-derived" can refer to chicken-derived, turkey-derived and / or quail-derived.

"Retroviral particles" or "transduced particles" refer to replication-deficient or replication-type viruses capable of transducing nonviral DNA or RNA into cells. In a particularly useful embodiment, the retroviral particles used to make transgenic birds according to the present invention are made as disclosed in US Pat. No. 7,524,626 issued April 28, 2009. .. The disclosure of the above patent is incorporated herein by reference in its entirety.

The terms "transformation," "transduction," and "transfection" all refer to the introduction of polynucleotides into avian blastoderm cells. The "tubule" is the portion of the fallopian tube that lies between the funnel and the isthmus and contains tubular gland cells that synthesize and secrete the egg white protein of the egg.

The term "transgene" refers to a heterologous nucleotide sequence inserted into the trigenome according to the present invention. An "introductory gene" is an exogenous coding sequence, an exogenous coding sequence linked to an exogenous promoter or other regulatory sequence, all nucleotide sequences between two retrovirus LTRs and / or retrovirus LTRs, and an introduction. It may specifically refer to a nucleotide sequence between the LTRs of the retrovirus used to introduce the gene.

The term "optimization" is used in the context of "optimization coding sequence", where the most commonly used codons for each particular amino acid found in the egg white proteins ovalbumin, lysozyme, ovomucoid and ovotransferase are , Used in the design of optimized human interferon-α2b (IFN-α2b) polynucleotide sequences inserted into the vectors of the invention. More specifically, the DNA sequence for optimized human IFN-α2b is based on codon usage optimized for female chicken transferrin, and chicken (Gal1us Gal1us) ovalbumin, lysozyme, ovomucoid and Wisconsin Package, Version 9.1 BACKTRANSLATE Program (Genetics Computer Group) by codon usage table collected from ovalbumin transferrin protein Made using Inc., Madison, Wis.). For example, the percentage of four codons used in the four egg white proteins for the amino acid alanine is 34% for GCU, 31% for GCC, 26% for GCA, and 8% for GCG. Therefore, GCU is used as the codon for most alanine in the optimized coding sequence. Vectors containing genes for optimizing human proteins are used to generate transgenic birds that express transgenic poultry-derived proteins in their tissues and eggs.

The term "subject" as used herein includes mammalian and non-mammalian animals. Examples of mammals include, but are not limited to, humans, chimpanzees, tailless monkeys, tailed monkeys, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, and the like. Be done.

As used herein, the term "therapeutically effective amount" refers to an improvement in the treatment, cure, prevention or alleviation of a disease, disorder or side effect, or disease or disorder as compared to a corresponding control not taking that amount. Refers to any amount of compound that results in a reduction in the rate of progression of. Also included within the scope of this term are amounts that are effective in enhancing normal physiology.

The terms "treat," "treat," or "treat" prophylactically and / or therapeutically alleviate, ameliorate, or alleviate the symptoms of a disease or condition, prevent further symptoms, and the underlying cause of the symptoms. Alleviate or prevent, suppress the disease or condition, prevent the development of the disease or condition, alleviate the disease or condition, regress the disease or condition, alleviate the condition caused by the disease or condition, or the disease or condition Refers to a method of stopping the symptoms of the condition.

LAL composition The present invention generally relates to compositions comprising enzymes useful in the treatment, eg, the treatment of lysosomal storage diseases. In one aspect, the invention relates to lysosomal storage disease enzymes such as LAL that have a glycosylation pattern that makes the molecule susceptible to internal translocation by a particular cell type. Also included in the present invention are recombinant human proteins containing isolated or purified forms of LAL. Isolation of lysosomal storage disease enzymes (such as LAL) can be performed by methodologies that are readily apparent to those skilled in the art of protein purification.

In one embodiment, the invention relates to a lysosomal storage disease enzyme, including LAL, that has, but is not limited to, the N-linked glycosylation pattern described herein.

In one aspect, the compositions disclosed herein comprise human LALs, and a significant proportion of human LALs undergo internal translocation by mannose-6-phosphate receptors on the cell surface, eg, on hepatocytes. Contains a mannose-6-phosphate glycan moiety that can act as a ligand for. In one embodiment, at least 30% of the LAL contained in the composition, eg, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. , At least 97% or at least 99% contain at least one mannose-6-phosphate moiety. The mannose-6-phosphate moiety is, for example, Asn of SEQ ID NO: 2.¹⁵, Asn⁵¹, Asn⁸⁰, Asn¹⁴⁰, Asn²⁵²And Asn³⁰⁰It can be seen on the N-glycan structure located at one or more residues selected from the group consisting of. Glycan structures containing a mannose-6-phosphate moiety include, for example, Gn and Hn shown in FIG.

The recombinant human LAL according to the invention comprises multiple N-linked carbohydrate chains (eg, about 5 or 6 carbohydrate chains). The N-linked glycosylation structures at each of the 5 or 6 sites are An, Bn, Cn, Dn, En, Fn, Gn, Hn, In, Jn, Kn, Ln, Mn, Nn and On shown in FIG. You can choose from one of them.

Mixtures of LAL molecules (eg, two or more LAL molecules such as the LAL molecules described in SEQ ID NOs: 2, 3, 4 and 19 can be present in the mixture) are also described herein. Some or all of the LAL molecules are structure An, structure Bn, structure Cn, structure Dn, structure En, structure Fn, structure Gn, structure Hn, structure In, structure Jn, structure Kn, structure Ln, structure Mn, structure. It has one or more glycosylated structures selected from Nn and structure On (Fig. 16). In one embodiment, a mixture of lysosomal acid lipase molecules is purified or isolated, eg, isolated from eggs produced in transgenic birds, or purified or isolated from egg whites produced in transgenic birds.

The present invention also includes individual LAL molecules containing structure An. The present invention also includes individual LAL molecules containing structure Bn. The present invention also includes individual LAL molecules containing structural Cn. The present invention also includes individual LAL molecules containing structural Dn. The present invention also includes individual LAL molecules containing structural En. The present invention also includes individual LAL molecules containing structural Fn. The present invention also includes individual LAL molecules containing structural Gn. The present invention also includes individual LAL molecules containing structural Hn. The present invention also includes individual LAL molecules containing structure In. The present invention also includes individual LAL molecules containing structural Jn. The present invention also includes individual LAL molecules containing structural Kn. The present invention also includes individual LAL molecules containing structural Ln. The present invention also includes individual LAL molecules containing structural Mn. The present invention also includes individual LAL molecules containing structural Nn. The present invention also includes individual LAL molecules containing structure On.

The N-linked oligosaccharides bound to human LAL according to the present invention are deficient in terminal sialic acid and galactose residues. That is, only a small number of N-linked oligosaccharide structures are sial-oxidized at the ends, and only a small number of galactose residues are present. In addition, terminal N-acetylglucosamine (GlcNAc) is widely present on the N-linked oligosaccharide structures of LALs described herein. Thus, LALs produced according to the present invention can be targeted against cells such as monocyte macrophages and Kupffer cells.

In one aspect of the invention, a composition of LAL that is substantially free of sialic acid is provided. In another aspect, the compositions disclosed herein contain recombinant human LAL and none of its N-glycan structures contain a sialic acid moiety that can interfere with the intracellular translocation of the enzyme into the cell. .. In one embodiment, 15% or less, eg, 10% or less, 5% or less, 2% or less, 1% or less of the LAL contained in the composition contains a sialic acid moiety in its N-glycan structure or is substantial. All LALs do not contain sialic acid moieties.

In another embodiment, about 95% or more of the N-linked oligosaccharides present on the individual LAL molecules of the invention are sialic acid free. In another embodiment, about 90% or more of the N-linked oligosaccharides present on the individual LAL molecules of the invention are sialic acid free. In another embodiment, about 80% or more of the N-linked oligosaccharides present on the individual LAL molecules of the invention are sialic acid free. In another embodiment, about 70% or more of the N-linked oligosaccharides present on the individual LAL molecules of the invention are sialic acid free.

In yet another embodiment, substantially all N-linked oligosaccharide structural types present on the LAL molecule of the invention are sialic acid free. In another embodiment, about 90% or more of the N-linked oligosaccharide structural types known to be bound to the LAL molecules of the invention are sialic acid free. For example, if there are 20 oligosaccharide structure types, then 18 or more structure types do not contain sialic acid. In another embodiment, about 80% or more of the N-linked oligosaccharide structural types known to be bound to the LAL molecules of the invention are sialic acid free. In another embodiment, about 70% or more of the N-linked oligosaccharide structural types known to be bound to the LAL molecule of the invention are sialic acid free. In another embodiment, about 60% or more of the N-linked oligosaccharide structural types known to be bound to the LAL molecules of the invention are sialic acid free. In another embodiment, about 50% or more of the N-linked oligosaccharide structural types known to be bound to the LAL molecules of the invention are sialic acid free.

According to one aspect of the invention, the LALs described herein contain high levels of terminal N-acetylglucosamine. In one aspect, about 95% or more of the N-linked oligosaccharides present on the individual LAL molecules of the invention contain terminal N-acetylglucosamine. In another embodiment, about 90% or more of the N-linked oligosaccharides present on the individual LAL molecules of the invention contain terminal N-acetylglucosamine. In another embodiment, about 80% or more of the N-linked oligosaccharides present on the individual LAL molecules of the invention contain terminal N-acetylglucosamine. In another embodiment, about 70% or more of the N-linked oligosaccharides present on the individual LAL molecules of the invention contain terminal N-acetylglucosamine. In another embodiment, about 60% or more of the N-linked oligosaccharides present on the individual LAL molecules of the invention contain terminal N-acetylglucosamine. In another embodiment, about 50% or more of the N-linked oligosaccharides present on the individual LAL molecules of the invention contain terminal N-acetylglucosamine.

In one embodiment, all N-linked oligosaccharide structural types present on the LAL molecule of the invention comprise terminal N-acetylglucosamine. In another embodiment, about 90% or more of the N-linked oligosaccharide structure types present on the LAL molecule of the present invention contain terminal N-acetylglucosamine. For example, if there are 20 oligosaccharide structure types, then 18 or more oligosaccharide structure types do not contain terminal N-acetylglucosamine. In another embodiment, about 80% or more of the N-linked oligosaccharide structure types present on the LAL molecule of the present invention contain terminal N-acetylglucosamine. In another embodiment, about 70% or more of the N-linked oligosaccharide structure types present on the LAL molecule of the present invention contain terminal N-acetylglucosamine. In another embodiment, about 60% or more of the N-linked oligosaccharide structure types present on the LAL molecule of the present invention contain terminal N-acetylglucosamine. In another embodiment, about 50% or more of the N-linked oligosaccharide structure types present on the LAL molecule of the present invention contain terminal N-acetylglucosamine.

In another aspect of the invention, the compositions disclosed herein contain human LALs, and a significant proportion of human LALs do not contain fucose moieties in any of their N-glycan structures. In one embodiment, 50% or less of the LAL contained in the composition, for example, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 2% or less, 1% or less. Contains a fucose moiety in its N-glycan structure, or virtually all LALs do not contain a fucose moiety.

In one embodiment, fucose is substantially absent on the N-linked oligosaccharide structure of LAL produced according to the present invention. In another embodiment, about 95% or more of the N-linked oligosaccharides present on the individual LAL molecules of the invention are fucose-free. In another embodiment, about 90% or more of the N-linked oligosaccharides present on the individual LAL molecules of the invention are fucose-free. In another embodiment, about 85% or more of the N-linked oligosaccharides present on the individual LAL molecules of the invention are fucose-free. In another embodiment, about 80% or more of the N-linked oligosaccharides present on the individual LAL molecules of the invention are fucose-free. In another embodiment, about 70% or more of the N-linked oligosaccharides present on the individual LAL molecules of the invention are fucose-free. In another embodiment, about 60% or more of the N-linked oligosaccharides present on the individual LAL molecules of the invention are fucose-free. In another embodiment, about 50% or more of the N-linked oligosaccharides present on the LAL molecule of the present invention are fucose-free.

In one embodiment, substantially all N-linked oligosaccharide structural types present on the LAL molecule of the present invention do not contain fucose. In another embodiment, about 95% or more of the N-linked oligosaccharide structure types present on the LAL molecule of the present invention are fucose-free. For example, if there are 20 oligosaccharide structure types, then 19 or more structure types do not contain fucose. In another embodiment, about 90% or more of the N-linked oligosaccharide structure types present on the LAL molecule of the present invention are fucose-free. In another embodiment, about 85% or more of the N-linked oligosaccharide structure types present on the LAL molecule of the present invention are fucose-free. In another embodiment, about 80% or more of the N-linked oligosaccharide structure types present on the LAL molecule of the present invention are fucose-free. In another embodiment, about 70% or more of the N-linked oligosaccharide structure types present on the LAL molecule of the present invention are fucose-free.

As mentioned above, there are many specific monosaccharides in the LAL molecule produced according to the present invention. The total monosaccharide species analyzed include fucose, N-acetylgalactosamine, N-acetylglucosamine, galactose, glucose, mannose, mannose-6-phosphate, N-acetylneuraminic acid and N-glycolylneuraminic acid. Can be mentioned. Fucose can be present between about 0% and about 1% of the total monosaccharide composition. N-Acetylgalactosamine can be present between about 0% and about 1% of the total monosaccharide composition. N-Acetylglucosamine can be present between about 35% and about 50% of the total monosaccharide composition. Galactose can be present between about 1-10% of the total monosaccharide composition. Glucose can be present at 0% of the total monosaccharide composition. Mannose can be present between about 32% and about 50% of the total monosaccharide composition. Mannose-6-phosphate can be present between about 1% and about 11% of the total monosaccharide composition.

In one embodiment, the LAL produced according to the present invention is xylose-free. Moreover, one aspect of the invention comprises a composition of LAL without O-linked glycosylation, as there is virtually no N-acetylgalactosamine (GalNac) in the LAL produced according to the invention.

LAL has six potential sites for N-linked glycosylation in its amino acid sequence, such as Asn in SEQ ID NO: 1.³⁶, Asn⁷², Asn¹⁰¹, Asn¹⁶¹, Asn²⁷³And Asn³²¹Have. Five of them, Asn³⁶, Asn¹⁰¹, Asn¹⁶¹, Asn²⁷³And Asn³²¹Is glycosylated, but Asn⁷²Can be non-glycosylated or substantially non-glycosylated (substantially non-glycosylated means Asn in a mixture of LAL molecules³⁶, Asn¹⁰¹, Asn¹⁶¹, Asn²⁷³And Asn³²¹Less than any of Asn⁷²Means that is glycosylated) (see Figure 17). Therefore, one aspect of the present invention is Asn.⁷²Is a composition of LAL that is non-glycosylated and / or substantially non-glycosylated in. Glycosylated Asn⁷²LAL having is within the scope of the present invention. The position of Asn described herein is based on the LAL amino acid sequence set forth in SEQ ID NO: 1. Asn numbering (ie, asparagine position) can vary by individual LAL molecule and is easy in other LAL molecules such as the LAL molecule whose amino acid sequence is described by SEQ ID NOs: 2, 3, 4 and 19. It will be clear to those skilled in the art that it can be determined.

The LAL produced according to the present invention contains an N-glycan structure containing a mixture of bi-branched, tri-branched and quaternary structures having N-acetylglucosamine, mannose and mannose-6-phosphate (M6P) as major sugars ( Figures 16 and 17). According to one aspect of the invention, at least Asn¹⁰¹, Asn¹⁶¹And Asn²⁷³There is an M6P-modified N-glycan in. Therefore, one embodiment of the present invention is Asn.¹⁰¹, Asn¹⁶¹Or Asn²⁷³Contains a composition of LAL having an M6P-modified N-glycan present in any one of the above. In yet another embodiment, the invention is Asn.²⁷³Contains a composition of LAL with M6P modified N-glycans present in. In another embodiment, the invention is Asn.¹⁰¹, Asn¹⁶¹Or Asn²⁷³Contains a composition of LAL having a monophosphorylated N-glycan (M6P) present in any one of the above. In yet another embodiment, the invention is Asn.¹⁶¹And Asn²⁷³Contains a composition of LAL with monophosphorylated N-glycans present in. In yet another embodiment, the invention is Asn.¹⁰¹And Asn²⁷³Contains a composition of LAL with monophosphorylated N-glycans present in. In one particular embodiment, the LAL produced according to the present invention is Asn.¹⁰¹May contain mannose diphosphate (bis-M6P).

LALs produced according to the present invention may contain reduced levels of galactose (eg, "Gal"). One aspect of the present invention is Asn.³⁶, Asn¹⁶¹Or Asn³²¹Contains a composition of LAL having terminal galactose in any one of the above. In yet another embodiment, the invention is Asn.³⁶And Asn¹⁶¹Contains a composition of LAL having terminal galactose in. In yet another embodiment, the invention is Asn.¹⁶¹And Asn³²¹Contains a composition of LAL having terminal galactose in. In yet another embodiment, the invention is Asn.³⁶And Asn³²¹Contains a composition of LAL having terminal galactose in. In yet another embodiment, the invention is Asn.³⁶, Asn¹⁶¹And Asn³²¹Contains a composition of LAL having terminal galactose in. In yet another embodiment, the invention comprises a composition of LAL without terminal galactose.

Various types of N-glycans were found at various N-linked glycosylation sites in LAL. The N-glycan structure contains a mixture of bi-branched, tri-branched and quaternary structures with N-acetylglucosamine, mannose and mannose-6-phosphate (M6P) as the major sugars. Specifically, in one embodiment of the invention, the LAL is the first N-linked glycosylation site (eg, Asn in SEQ ID NO: 1).³⁶) Contains an N-glycan structure selected from GlcNAc4Man3GlcNAc2 or Gal1GlcNAc4Man3GlcNAc2. In another embodiment, the LAL is a second N-linked glycosylation site (eg, Asn in SEQ ID NO: 1).⁷²), Glycosylation-free or substantially non-glycosylation. In yet another embodiment, the LAL is a third N-linked glycosylation site (eg, Asn in SEQ ID NO: 1).¹⁰¹) Includes Phos2Man7GlcNAc2. In yet another embodiment, the LAL is the fourth N-linked glycosylation site (eg, Asn in SEQ ID NO: 1).¹⁶¹) Contains Phos1Man6GlcNAc2, GlcNAc1Phos1Man6GlcNAc2, Man3GlcNAc2, GlcNAc2Man3GlcNAc2, GlcNAc3Man3GlcNAc2, GlcNAc4Man3GlcNAc2 or Gal1GlcNAc4Man3GlcNAc2. In yet another embodiment, the LAL is the fifth N-linked glycosylation site (eg, Asn in SEQ ID NO: 1).²⁷³) Includes an N-glycan structure selected from Man7GlcNAc2, Man8GlcNAc2, Man9GlcNAc2, Phos1Man8GlcNAc2 or Phos1Man9GlcNAc2. In yet another embodiment, the LAL is the sixth N-linked glycosylation site (eg, Asn in SEQ ID NO: 1).³²¹), GlcNAc2Man3GlcNAc2, GlcNAc3Man3GlcNAc2, GlcNAc4Man3GlcNAc2, Gal1GlcNAc4Man3GlcNAc2, GlcNAc5Man3GlcNAc2, Gal1GlcNAc5Man3GlcNAc2, Gal1GlcNAc5Man3GlcNAc2, Gal1GlcNAc5Man3GlcNAc2, Gal1GlcNAc5Man3GlcNAc2, Gal1GlcNAc5Man3GlcNAc2, Gal1GlcNAc5Man3GlcNAc2, Gal1GlcNAc5Man3GlcNAc2

According to one particular aspect of the invention, the composition of LAL is the Asn of SEQ ID NO: 1.³⁶, Asn⁷², Asn¹⁰¹, Asn¹⁶¹, Asn²⁷³And Asn³²¹It contains a glycosylated LAL at (or the corresponding asparagine residue in SEQ ID NOs: 2, 3, 4 and 19) and one N-glycan is at the Asn position specified as shown below: a) Asn³⁶To GlcNAc4Man3GlcNAc2 or There is Gal1GlcNAc4Man3GlcNAc2; b) Asn⁷²Without glycosylation; c) Asn¹⁰¹Has Phos2Man7GlcNAc2 in d) Asn¹⁶¹To Phos1Man6GlcNAc2, GlcNAc1Phos1Man6GlcNAc2, Man3GlcNAc2, GlcNAc2Man3GlcNAc2, GlcNAc3Man3GlcNAc2, GlcNAc4Man3 GlcNAc2 or There is Gal1GlcNAc4Man3GlcNAc2; e) Asn²⁷³To Man7GlcNAc2, Man8GlcNAc2, Man9GlcNAc2, Phos1Man8GlcNAc2 or There is Phos1Man9GlcNAc2; f) Asn³²¹To GlcNAc2Man3GlcNAc2, GlcNAc3Man3GlcNAc2, GlcNAc4Man3GlcNAc2, Gal1GlcNAc4Man3GlcNAc2, GlcNAc5Man3GlcNAc2, Gal1GlcNAc5Man3GlcNAc2, GlcNAc6Man3 GlcNAc2 or Gal1GlcNAc6Man3GlcNAc2 There is, during the ceremony, Man = Mannose GlcNAc = N-acetylglucosamine Phos = Phosphate Ester Gal = galactose Is.

In one embodiment, Asn in LAL produced according to the present invention.³⁶, Asn¹⁶¹Or Asn³²¹In any one of the above, Gal1GlcNAc4Man3GlcNAc2 is found as a glycan component. In one particular embodiment, Asn³⁶, Asn¹⁶¹And Asn³²¹Gal1GlcNAc4Man3GlcNAc2 can be seen as a glycan component of.

In the LAL of the present invention, Asn¹⁰¹And Asn²⁷³However, a high mannose type (MAN6-MAN10 described in the present specification) having about 6 to about 10 mannose molecules as a main component is shown. Therefore, one aspect of the present invention is Asn.¹⁰¹Or Asn²⁷³Contains a composition of LAL having a high mannose structure in. In another embodiment, the LAL composition of the present invention is Asn.¹⁰¹Or Asn²⁷³May contain an N-glycan structure with at least 6 mannoses. In another embodiment, the composition of LAL is Asn.¹⁰¹Or Asn²⁷³Contains N-glycans with 7, 8 or 9 mannoses. In yet another embodiment, the invention is Asn.¹⁰¹And Asn²⁷³Contains a composition of LAL having 7, 8 or 9 mannoses in. In yet another embodiment, the invention is Asn.¹⁰¹And Asn²⁷³Contains a composition of LAL having 7, 8 or 9 mannoses in which one of the mannoses is phosphorylated.

The glycosylation site and the numbers associated with Asn are based on the amino acid sequence of LAL described in SEQ ID NO: 1, and the corresponding Asn numbering may vary by LAL molecule, but with respect to SEQ ID NO: 1. It should be understood that the glycosylation profile also applies to the LAL molecules described in SEQ ID NOs: 2, 3, 4 and 19. For example, Asn in SEQ ID NO: 1³⁶Is Asn of SEQ ID NO: 2¹⁵, Asn in SEQ ID NO: 3¹³, Asn of SEQ ID NO: 4¹⁰And Asn of SEQ ID NO: 19⁹Corresponds to. Asn of SEQ ID NO: 1⁷²Is Asn of SEQ ID NO: 2⁵¹, Asn in SEQ ID NO: 3⁴⁹, Asn of SEQ ID NO: 4⁴⁶And Asn of SEQ ID NO: 19⁴⁵Corresponds to. Asn of SEQ ID NO: 1¹⁰¹Is Asn of SEQ ID NO: 2⁸⁰, Asn in SEQ ID NO: 3⁷⁸, Asn of SEQ ID NO: 4⁷⁵And Asn of SEQ ID NO: 19⁷⁴Corresponds to. Asn of SEQ ID NO: 1¹⁶¹Is Asn of SEQ ID NO: 2¹⁴⁰, Asn in SEQ ID NO: 3¹³⁸, Asn of SEQ ID NO: 4¹³⁵And Asn of SEQ ID NO: 19¹³⁴Corresponds to. SEQ ID NO: 1²⁷³Is Asn of SEQ ID NO: 2²⁵², Asn in SEQ ID NO: 3²⁵⁰, Asn of SEQ ID NO: 4²⁴⁷And Asn of SEQ ID NO: 19²⁴⁶Corresponds to. Asn of SEQ ID NO: 1³²¹Is Asn of SEQ ID NO: 2³⁰⁰, Asn in SEQ ID NO: 3²⁹⁸, Asn of SEQ ID NO: 4²⁹⁵And Asn of SEQ ID NO: 19²⁹⁴Corresponds to.

For example, in one embodiment, LAL is Asn of SEQ ID NO: 2.¹⁵, Asn⁵¹, Asn⁸⁰, Asn¹⁴⁰, Asn²⁵²And Asn³⁰⁰It is N-linked glycosylated at at least one position selected from the group consisting of. In another embodiment, the LAL is Asn of SEQ ID NO: 2.¹⁵, Asn⁸⁰, Asn¹⁴⁰, Asn²⁵²And Asn³⁰⁰Is N-linked glycosylated in. In yet another embodiment, the N-glycan structure of LAL of SEQ ID NO: 2 does not have xylose, but the N-glycan structure of 15%, 10%, 5% or less than 1% contains sialic acid; 50%. , 40%, 30%, 20%, 10%, 5% or less than 1% N-glycan structure contains fucose; at least 30%, 50%, 60%, 70%, 80%, 90% and 95% N-glycan structure contains phosphorylated mannose (M6P).

In one embodiment, the LAL is Asn of SEQ ID NO: 3.¹³, Asn⁴⁹, Asn⁷⁸, Asn¹³⁸, Asn²⁵⁰And Asn²⁹⁸It is N-linked glycosylated at at least one position selected from the group consisting of. In another embodiment, the LAL is Asn of SEQ ID NO: 3.¹³, Asn⁷⁸, Asn¹³⁸, Asn²⁵⁰And Asn²⁹⁸Is N-linked glycosylated in. In yet another embodiment, the N-glycan structure of LAL of SEQ ID NO: 3 does not have xylose, but the N-glycan structure of 15%, 10%, 5% or less than 1% contains sialic acid; 50%. , 40%, 30%, 20%, 10%, 5% or less than 1% N-glycan structure contains fucose; at least 30%, 50%, 60%, 70%, 80%, 90% and 95% N-glycan structure contains phosphorylated mannose (M6P).

In one embodiment, the LAL is Asn of SEQ ID NO: 4.¹⁰, Asn⁴⁶, Asn⁷⁵, Asn¹³⁵, Asn²⁴⁷And Asn²⁹⁵It is N-linked glycosylated at at least one position selected from the group consisting of. In another embodiment, the LAL is Asn of SEQ ID NO: 4.¹⁰, Asn⁷⁵, Asn¹³⁵, Asn²⁴⁷And Asn²⁹⁵Is N-linked glycosylated in. In yet another embodiment, the N-glycan structure of LAL of SEQ ID NO: 4 does not have xylose, but the N-glycan structure of 15%, 10%, 5% or less than 1% contains sialic acid; 50%. , 40%, 30%, 20%, 10%, 5% or less than 1% N-glycan structure contains fucose; at least 30%, 50%, 60%, 70%, 80%, 90% and 95% N-glycan structure contains phosphorylated mannose (M6P).

In one embodiment, the LAL is Asn of SEQ ID NO: 19.⁹, Asn⁴⁵, Asn⁷⁴, Asn¹³⁴, Asn²⁴⁶And Asn²⁹⁴It is N-linked glycosylated at at least one position selected from the group consisting of. In another embodiment, LAL is Asn of SEQ ID NO: 19.⁹, Asn⁷⁴, Asn¹³⁴, Asn²⁴⁶And Asn²⁹⁴Is N-linked glycosylated in. In yet another embodiment, the N-glycan structure of LAL of SEQ ID NO: 4 does not have xylose, but the N-glycan structure of 15%, 10%, 5% or less than 1% contains sialic acid; 50%. , 40%, 30%, 20%, 10%, 5% or less than 1% N-glycan structure contains fucose; at least 30%, 50%, 60%, 70%, 80%, 90% and 95% N-glycan structure contains phosphorylated mannose (M6P).

The compositions according to the invention can be prepared by the use of transgenic birds, transgenic fish, transgenic mammals such as transgenic goats, or in transgenic plants such as tobacco and duckweed (Lemna minor) and certain types of cultured cells. It can be produced in many ways, including.

The present invention also contemplates compositions containing pegged LAL. The LAL enzymes described herein can be pegged, for example, as disclosed in US Patent Application Publication No. 20070092486 published April 26, 2007. The disclosure of the above patent is incorporated herein by reference in its entirety.

In one embodiment, the induced glycosylation pattern is obtained from, for example, an expression-specific expression system derived from avian oviduct cells, such as tubular gland cells. For example, the glycosylation patterns disclosed herein have been shown to be present on lysosomal storage disease enzymes produced in oviduct cells of chickens such as chickens according to the present invention.

Proteins produced according to the present invention can be purified from egg white by any useful method, such as those apparent to those skilled in the art of protein purification. For example, human LAL (hLAL) produced in transgenic birds according to the present invention can be purified from egg white by methods apparent to those skilled in the art of protein purification. An example of a purification protocol for LAL present in egg white is described in the Examples.

The present invention comprises eggs and egg whites containing the lysosomal acid lipase molecules of the invention comprising one or more of the glycosylation structures disclosed herein, as well as eggs-laying and egg-white-producing birds (eg, chickens, quail butterflies). And quail).

LAL expression in birds Proteins for which the addition of mannose-6-phosphate is effective (eg, increased efficacy is obtained) by stably introducing an exogenous nucleic acid sequence into the trigenome, such as lysosomal acid lipase (LAL) and the present specification. Vectors and methods for expressing desired proteins, such as lysosomal enzymes, including but not limited to other proteins specifically disclosed herein, are disclosed herein. Specifically, a transgenic bird that expresses an exogenous sequence in the oviduct and accumulates an exogenous protein such as a pharmaceutical protein in the egg is prepared. Bird eggs containing such foreign proteins are also described herein. Also disclosed herein are novel forms of LAL that are efficiently expressed in the oviduct of transgenic birds and accumulated in the eggs of the birds.

One aspect of the invention relates to LAL, a composition containing LAL molecules produced according to the invention. In a particularly effective embodiment, the LAL is purified or isolated. For example, LAL has been extracted from the contents of hard-shelled eggs laid by transgenic birds. In one particularly effective embodiment, the LAL is a human LAL. In one embodiment, the LALs of the present invention have a glycosylation pattern derived from the LALs produced in avian oviduct cells. For example, the composition may contain a mixture of LAL molecules produced in chickens, eg chickens, according to the present invention and isolated from egg white. In one effective embodiment, the LAL-containing composition is a pharmaceutical formulation.

In one aspect, the invention relates to a composition containing an isolated LAL molecule, eg, a human LAL molecule, which is produced in a bird carrying a transgene encoding LAL. In one embodiment, LAL is produced in the oviduct cells (eg, tubular gland cells) of a transgenic bird (eg, transgenic chicken) and the LAL is isolated from the egg white of the transgenic bird. In one embodiment, LAL is glycosylated within the oviductal cells (eg, tubular gland cells) of birds, such as chickens.

In another aspect, there is provided a method of producing an exogenous protein, such as LAL, in a particular tissue of a bird, such as a lysosomal storage disease enzyme. Such exogenous proteins can be expressed in avian fallopian tubes, blood and / or other cells and tissues. In one embodiment, for example, in the blastocyst near stage X, the protein of interest is expressed in the tubular gland cells of the oviductal tube, secreted into the lumen and accumulated in the egg white of the hard-shelled egg. Transgenes are introduced to produce transgenic birds. The transgenic bird thus produced can carry the transgene in its germ line and can stably transmit the exogenous transgene to its offspring by the Mendelian method.

The present invention includes a method of producing an exogenous protein such as LAL in a bird oviduct. The method comprises a coding sequence and a promoter operably linked to the coding sequence and may include a first step of providing a vector in which the promoter can result in nucleic acid expression within the avian oviduct. Transgenic cells and / or tissues can be generated, where the vector is introduced into freshly isolated, cultured, or intraembryo avian blastoderm cells, where the vector sequence is trigenome. To be inserted inside. From the transgenic cells and / or tissues, mature transgenic birds expressing exogenous proteins such as LAL in the oviduct can be obtained.

In one aspect of the invention, transgenic birds are transduced by transduction of germline lobe cells using replication-deficient or replication-type retroviral particles carrying a transgene between the 5'LTR and 3'LTR of the retroviral vector. Is produced. For example, a tricholemic virus (ALV) retroviral vector or a murine leukemia virus (MLV) retroviral vector containing a modified pNLB plasmid containing a foreign gene inserted downstream of a segment of the promoter region can be used. An RNA copy of the modified retroviral vector packaged within the viral particles can be used to infect the scutellum lobes that become transgenic birds.

In another aspect of the invention, there is provided a vector comprising a coding sequence and a promoter, which are in a directional and positional relationship such that the coding sequence is expressed in the avian oviduct. Such vectors include, but are not limited to, a tricholemia virus (ALV) retroviral vector, a murine leukemia virus (MLV) retroviral vector and a lentiviral vector. Further, the vector may be a nucleic acid sequence containing the LTR of a tricholemia virus (ALV) retroviral vector, a murine leukemia virus (MLV) retroviral vector or a lentiviral vector. The promoter is sufficient to result in the expression of the coding sequence within the avian oviduct. The coding sequence encodes an exogenous protein that accumulates in the egg white of hard-shelled eggs. Thus, the coding sequence encodes a protein from transgenic poultry, such as an exogenous protein such as lysosomal acid lipase (TPD LAL) from transgenic poultry.

In one embodiment, the vector used in the method of the invention comprises a promoter particularly suitable for expression of foreign proteins in birds and avian eggs. Therefore, expression of an exogenous coding sequence can occur in the oviduct and blood of transgenic birds and in the egg white of transgenic birds' eggs. Promoters include cytomegalovirus (CMV) promoter, laus sarcoma virus (RSV) promoter, β-actin promoter (eg, chicken β-actin promoter), mouse leukemia virus (MLV) promoter, mouse mammary tumor virus (MMTV) promoter. , Ovoalbumin promoter, lysoteam promoter, conalbumin promoter, ovomucoid promoter, ovomucin promoter and ovotransferase promoter, but are not limited thereto. Optionally, the promoter may be a segment of at least one promoter region, such as the ovalbumin, lysozyme, conalbumin, ovomucoid, ovomucin and ovotransferrin promoter regions. In one embodiment, the promoter is one or more combinations or fusions of promoters such as, for example, ovalbumin, lysozyme, conalbumin, ovomucoid, ovomucin and ovotransferrin promoters, or fusions of parts of one or more promoters. ..

In one embodiment, the vector comprises a signal peptide coding sequence that, upon intracellular translation, into the egg white of a hard shell egg of an exogenous protein such as human LAL expressed by the vector. It is operably linked to the coding sequence so that the signal peptide directs the secretion of.

In one aspect of the invention, a coding sequence for an exogenous protein produced as disclosed herein is provided, where the coding sequence is codon-optimized for expression in a chicken, eg chicken. .. Codon optimization can be determined from the codon usage frequency of at least one, preferably two or more proteins expressed in avian cells (eg, chicken cells). For example, codon usage can be determined from the nucleic acid sequences encoding the chicken proteins ovalbumin, lysozyme, ovomucin and ovotransferrin. For example, the DNA coding sequence of an exogenous protein can be found in the Wisconsin Package, Version 9.1 BACKTRANSLATE® program, with a codon usage table collected from chicken (Gal1us Gal1us) ovalbumin, lysozyme, ovomucoid and ovotransferrin proteins. Can be codon-optimized using (Genetics Computer Group Inc., Madison, Wis.).

An important aspect of the present invention relates to a chicken hard shell egg (eg, chicken hard shell egg) containing an exogenous peptide or protein, including but not particularly limited, human LAL. Exotic peptides or proteins such as human LAL can be encoded by transgenes in transgenic birds. Often, exogenous peptides or proteins (eg, LAL) are glycosylated. The protein can be present in any useful amount. In one embodiment, the protein is present in an amount ranging from about 0.01 μg per hard shell egg to about 1 gram per hard shell egg. In another embodiment, the protein is present in an amount ranging from about 1 μg per hard shell egg to about 1 gram per hard shell egg. For example, the protein may be present in an amount ranging from about 10 μg per hard shell egg to about 1 gram per hard shell egg (eg, from about 10 μg per hard shell egg to about 400 milligrams per hard shell egg). ..

In one embodiment, the exogenous protein of the invention is present in the egg white of an egg. In one embodiment, the protein is present in an amount ranging from about 1 ng per milliliter of egg white to about 0.2 grams per milliliter of egg white. For example, protein can be present in an amount ranging from about 0.1 μg per milliliter of egg white to about 0.2 grams per milliliter of egg white (eg, protein is an amount ranging from about 1 μg per milliliter of egg white to about 100 milligrams per milliliter of egg white). In one embodiment, the protein is present in an amount ranging from about 1 μg per milliliter of egg white to about 50 milligrams per milliliter of egg white. For example, the protein is present in an amount ranging from about 1 μg per milliliter of egg white to about 1 milliliter per milliliter of egg white. It can be present in an amount in the range of 10 milligrams (eg, protein can be present in an amount ranging from about 1 μg per milliliter of egg white to about 1 milligram per milliliter of egg white). It is present in an amount greater than 0.1 μg. In one embodiment, the protein is present in an amount greater than 0.5 μg per milliliter of egg white. In one embodiment, the protein is present in an amount greater than 1 μg per milliliter of egg white. In morphology, protein is present in an amount greater than 1.5 μg per milliliter of egg white.

The birds of the invention that produce the exogenous proteins disclosed herein (eg, LAL) that originated from vector-introduced blastocysts are of the G0 generation and are sometimes referred to as "creation initiators." .. Primordial birds are usually chimeric with respect to each transgene inserted. That is, only some cells of the G0 transgenic bird carry the transgene (s). The G0 generation is also usually hemizygous for the transgene (s). Mating the G0 generation with non-transgenic animals yields G1 transgenic progeny that are also hemizygous for the transgene and carry the transgene (s) substantially in all cells of the bird. obtain. G1 hemizygous offspring can be crossed with non -transgenic animals to produce G2 hemizygous offspring, and G1 hemizygous offspring can be crossed with each other to produce G2 offspring that are homozygous for the transgene. Virtually all avian cells that are positive for a transgene from G1 offspring carry the transgene (s). In one embodiment, hemizygous G2 offspring from the same strain can be crossed to obtain homozygous G3 offspring for the transgene. In one embodiment, hemizygous G0 or G1 individuals are mated with each other, for example, to produce homozygous G1 progeny carrying two copies of the transgene (s) in each cell of the individual. can do. These are merely examples of certain effective mating methods, and the present invention contemplates the use of any effective mating method, such as methods known to those of skill in the art.

In one embodiment, the invention provides an isolated LAL. That is, the LAL contained in the composition can be an isolated LAL. For example, LAL can be isolated from egg white. The isolated LAL can be a LAL molecule having various glycosylation structures within the LAL molecule.

A transgenic chicken, a transgenic citrus, which introduces a transgene into avian blastocyst cells by the method of the present invention and carries the transgene in a genetic material of a reproductive system tissue to produce the protein of the present invention. Transgenic quail and birds of other species can be produced. Blastocyst cells are usually stage VII-XII cells or equivalent cells, and in one embodiment, cells near stage X.

Several vectors are described herein that are useful in practicing the methods of the invention. In one embodiment, both the vector coding sequence and the promoter are located between the 5'LTR and 3'LTR prior to introduction into blastocyst cells. In one embodiment, the vector is a retrovirus and both the coding sequence and the promoter are located between the 5'LTR and 3'LTR of the retroviral vector. In one useful embodiment, the LTR or retroviral vector is derived from trileukemia virus (ALV), murine leukemia virus (MLV) or lentivirus.

In one embodiment, the vector used to transfect blastocyst cells to result in stable insertion into the lysosome encodes a coding sequence and a promoter within the tubular gland cells of the avian oviduct tube. Contains in a directional and positional relationship such that is expressed, where exogenous proteins such as lysosomal enzymes (eg, LAL) are accumulated in the egg white of hard-shelled eggs.

The promoter may optionally be a segment of the ovalbumin promoter region large enough to direct the expression of the coding sequence within tubular gland cells. Truncation of the ovalbumin promoter and / or aggregation of key regulatory elements of the ovalbumin promoter allows the promoter to retain the sequences required for expression in tubular gland cells of the oviductal tube and to be readily integrated into the vector. It is within the scope of the present invention to make it small enough. In one embodiment, a segment of the ovalbumin promoter region may be used. This segment contains the 5'adjacent region of the ovalbumin gene.

In addition, the promoter may be a promoter that is mostly, but not all, specific to the tube, such as the lysozyme promoter. The promoter may also be a mouse mammary tumor virus (MMTV) promoter. Alternatively, the promoter may be a constitutive promoter (eg, cytomegalovirus (CMV) promoter, Raus sarcoma virus (RSV) promoter, murine leukemia virus (MLV) promoter, etc.). In one embodiment, the promoters are cytomegalovirus (CMV) promoter, MDOT promoter, Raus sarcoma virus (RSV) promoter, murine leukemia virus (MLV) promoter, mouse mammary tumor virus (MMTV) promoter, ovoalbumin promoter, lysoteam promoter, Conalbumin promoter, ovomucoid promoter, ovomucin promoter and / or ovotransferase promoter. Optionally, the promoter may be at least one segment of the promoter region, such as a segment of the ovalbumin, lysozyme, conalbumin, ovomucoid, ovomucin and ovotransferrin promoter regions.

One method of transfecting blastocyst cells is to deliver the vector into the blastocyst cells using a packaged retroviral vector for integration into the trigenome.

Retroviruses useful for randomly introducing a transgene into the trigenome are replication-deficient murine leukemia virus (ALV), replication-deficient murine leukemia virus (MLV) or lentivirus. In one embodiment, the pNLB vector is modified by inserting the ovalbumin promoter and one or more exogenous genes between the 5'and 3'long terminal repeats (LTRs) of the retroviral genome. The present invention contemplates that any coding sequence downstream of a promoter active in tubular gland cells can be expressed in tubular gland cells. For example, the ovalbumin promoter drives the expression of the ovalbumin protein and is active in the tubular gland cells of the oviduct, so it can be expressed in the tubular gland cells of the oviductal tube.

In addition, any vector described herein may optionally contain a coding sequence encoding a signal peptide that directs the secretion of the protein expressed by the coding sequence of the vector from the tubular gland cells of the oviduct. This aspect effectively expands the range of exogenous proteins that can be accumulated in avian eggs using the methods described herein. Where the foreign protein would not be secreted without it, the vector containing the coding sequence is modified to include a DNA sequence containing about 60 bp encoding the signal peptide from the lysozyme gene. The DNA sequence encoding the signal peptide is inserted into the vector so that it is located at the N-terminus of the protein encoded by the DNA.

Another aspect of the invention is the use of an internal ribosome entry site (IRES) element in any vector of the invention that allows translation of two or more proteins from dyscistronic or polycistronic mRNAs. including. By fusing the IRES unit to the 5'end of one or more additional coding sequences and then inserting it at the end of the original coding sequence in the vector, the coding sequences will be separated from each other by the IRES.

In one embodiment, an IRES can be used to facilitate post-translational modification of a product because one coding sequence can encode an enzyme capable of modifying the product of another coding sequence. For example, the first coding sequence encodes collagen, which can be hydroxylated and activated by the enzyme encoded by the second coding sequence, where IRES is as understood in the art. To use.

In another aspect, a 3'untranslated region (3'UTR) is added to the coding sequence of the vector used in any of the methods of the invention to stabilize the RNA produced. When adding the 3'UTR to a retroviral vector, the directions of the promoter, gene X and 3'UTR in the construct must be reversed so that the addition of the 3'UTR does not interfere with the transcription of full-length genomic RNA. .. In one embodiment, the 3'UTR can be the 3'UTR of the ovalbumin or lysozyme gene, or any 3'UTR that functions within the intracellular cells, i.e. the late SV40 region.

In one embodiment, a constitutive promoter is used to express the transgene coding sequence in birds. In this case, expression is not limited to the tube, but also occurs in other tissues within the bird (eg, blood). The use of such transgenes, including constitutive promoters and coding sequences, is particularly suitable for causing or driving protein expression in the oviduct and subsequent secretion of the protein into the egg.

Particles for transduction (ie, transduction particles) are made for the vector and titrated to determine the appropriate concentration that can be used for injection into the embryo. A bird's egg is perforated and transduced particles are injected into the egg according to Speksnijder's procedure (US Pat. No. 5,897,998, the entire content of which is incorporated herein by reference). Approximately 21 days after infusion, eggs hatch and male birds are screened for mating. DNA is extracted from a sample of male chicken sperm to screen for G0 male chickens that contain the transgene in their sperm. G0 male chickens containing the highest levels of transgene in sperm samples are mated with non-transgenic female chickens by artificial insemination. Blood DNA samples are screened for the presence of transgene. Transgenic male chicken sera are tested for the presence or absence of exogenous proteins. Once the exogenous protein is identified, the sperm of the transgenic male chicken is used for artificial insemination of non-transgenic female chickens. As a result, a certain percentage of progeny carry the transgene (eg, greater than 50%). If an exogenous protein is present in an egg produced according to the present invention, the protein may be isolated. You may also test the biological activity of the protein.

The method of the present invention, which provides the production of an exogenous protein in the avian oviduct and the production of an egg containing the exogenous protein, provides a suitable vector and subsequently introduces the vector into scutellum cells. It involves a further step so that the vector is integrated into the trigenome. The next step is to obtain mature transgenic birds from the transgenic blastocysts produced in the previous step. Mature transgenic birds can be obtained from cells of blastoderm embryos in which the vector has been directly transfected or transduced into the embryo. The resulting embryos are grown and the chickens mature.

Transgenic birds made from blastocyst cells are known as wound initiators. Some of the wound initiators carry the transgene in the tubular gland cells of the oviductal tube. These birds express the exogenous protein encoded by the transgene in the oviduct. The exogenous protein can also be expressed in other tissues (eg, blood) in addition to the fallopian tubes. If the exogenous protein contains the appropriate signal sequence (s), the protein is secreted into the lumen of the fallopian tubes and into the egg white of the egg.

Among the wound initiators are germline wound initiators. A germline wound initiator is a wound initiator that can carry a transgene in a genetic material of a germline tissue and can also carry a transgene in a tubular gland cell of the ovary tube tube that expresses an exogenous protein. Therefore, according to the present invention, a transgenic bird may have tubular gland cells expressing an exogenous protein, and progeny of the transgenic bird may also have oviduct tube tubular gland cells expressing an exogenous protein. Alternatively, its offspring express a phenotype determined by the expression of the exogenous gene within a particular tissue (s) of the bird. In one embodiment, the transgenic bird is a chicken or turkey.

Pharmaceutical composition and treatment Although it is possible to administer the therapeutic protein produced as described herein in an unprocessed form for therapeutic use, it is preferred to administer the therapeutic protein as part of a pharmaceutical formulation. .. Thus, a glycosylation therapeutic protein such as poultry-derived LAL or a pharmaceutically acceptable derivative thereof, with one or more pharmaceutically acceptable carriers and optionally other therapeutic and / or prophylactic components. Further provided are pharmaceutical formulations to be included, as well as methods of administration of such pharmaceutical formulations. The carrier (s) must be "acceptable" in the sense that it is compatible with the other ingredients of the formulation and is not harmful to the recipient. Treatment of patients using the pharmaceutical compositions of the present invention (eg, amount of pharmaceutical protein to be administered, frequency of administration, and length of treatment period) uses standard methods known to physicians skilled in the art. Can be decided.

See, for example, Remington's Pharmaceutical Sciences, 14th Edition, Mack Publishing Co., Easton, Pa. To be formulated). The carrier may include a diluent. In one embodiment, the pharmaceutical carrier can be in liquid form and the recombinant human LAL can be in solution form. The pharmaceutical carrier can be wax, fat or alcohol. In one embodiment, the wax or fat-based carrier is ester-free. In another embodiment, the pharmaceutically acceptable carrier can be a powder, lyophilized powder or solid in tablet form. In one embodiment, the carrier may comprise liposomes or microcapsules.

Pharmaceutical formulations include those suitable for administration by injection, including intramuscular, subcutaneous and intravenous administration. Pharmaceutical formulations include those suitable for oral, transrectal, nasal, topical (including buccal and sublingual), vaginal or parenteral. In addition, pharmaceutical preparations include those suitable for administration by inhalation or inhalation. The formulation can be conveniently provided in individual dosage units, if desired, and can be prepared by any method known in the pharmaceutical art. The method for producing a pharmaceutical formulation usually comprises mixing the therapeutic protein with a liquid carrier, a micronized solid carrier, or both, and then, if necessary, shaping the product into the desired formulation.

Pharmaceutical formulations suitable for oral administration are conveniently provided as individual units such as capsules, cashiers or tablets, each containing a predetermined amount of the active ingredient; powder or granules; liquid; suspension; or emulsion. obtain. The active ingredient may be provided as a large pill, lick or paste. Tablets and capsules for oral administration may contain conventional additives such as binders, excipients, lubricants, disintegrants or wetting agents. Tablets can be coated by methods known in the art. Oral liquid formulations may be in the form of, for example, aqueous or oily suspensions, liquids, emulsions, syrups or elixirs, and may be provided as a dry product composed of water or other suitable solvent prior to use. Such liquid formulations may contain conventional additives such as suspending agents, emulsifiers, non-aqueous solvents (which may include edible oils) or preservatives.

LAL may also be prepared for parenteral administration (eg, by injection, eg bolus injection or continuous infusion), in ampoules, prefilled syringes, in unit dose form in small infusions, or with preservatives added. Can be provided in a multi-dose container. Therapeutic proteins can be injected, for example, by subcutaneous injection, intramuscular injection and intravenous injection or injection.

The LAL can be in the form of suspensions, liquids or emulsions in oily or aqueous solvents and may contain pharmaceutical agents such as suspending agents, stabilizers and / or dispersants. It is also contemplated that the Therapeutic protein may be in powder form to be composed of a suitable solvent prior to use, such as sterile pyrogen water, obtained by sterile isolation from sterile solids or lyophilization from solution. Will be done.

For intravenous infusion or injection, the LAL produced according to the present invention can be formulated as an aqueous suspension or solution. Suitable additives for intravenous injectable or injectable formulations may include one of trisodium citrate dehydrate, citric acid and human serum albumin. The pharmaceutical product may also contain other suitable additives known in the art for use in other products for impaired lysosomal accumulation. The pH of LAL produced according to the present invention is maintained between about 5.6 and about 6.2. Preferably, the pH of the LAL preparation is maintained at 5.9 ± 0.2.

For topical administration to the skin, the therapeutic proteins of the invention produced according to the invention can be formulated as ointments, creams or lotions, or as transdermal patches. Ointments and creams can be formulated with an aqueous or oily base, for example, with the addition of suitable thickeners and / or gelling agents. Lotions can be formulated with an aqueous or oily base and may also contain one or more emulsifiers, stabilizers, dispersants, suspending agents, thickeners or colorants.

Suitable formulations for topical oral administration include flavored bases, usually lozenges containing active ingredients in sucrose and gum arabic or tragacant; inert groups such as gelatin and glycerin or sucrose and gum arabic. Examples include incense tablets containing the active ingredient in the agent; and an oral cleaning agent containing the active ingredient in a suitable liquid carrier.

The pharmaceutical preparation suitable for transrectal administration in which the carrier is a solid is most preferably a unit dose suppository. Suitable carriers include cocoa butter and other materials commonly used in the art, and suppositories are a template after mixing with a softened or dissolved carrier (s) of the active compound. It may be convenient to put it in a container, cool it, mold it, and form it.

Formulations suitable for vaginal administration contain, in addition to the active ingredient, carriers known in the art to be suitable, vaginal suppositories, tampons, creams, gels, pastes, foams. It may be provided as an agent or a spray agent.

For intranasal administration, the therapeutic proteins of the invention can be used as liquid sprays or dispersed powders, or in the form of drops. The drop may be formulated with an aqueous or non-aqueous base that also contains one or more dispersants, solubilizers or suspending agents. Liquid sprays are conveniently delivered from pressure packs.

For administration by inhalation, the therapeutic proteins according to the invention can be conveniently delivered from a blower, atomizer or pressure pack, or other means convenient for delivering aerosol sprays. Pressurized packs may contain suitable propellants such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gases. For pressurized aerosols, the unit of administration may be determined by providing a valve for delivering the measured amount.

For administration by inhalation or blowing, the Therapeutic protein according to the invention may take the form of a dry powder composition, eg, a mixed powder of a compound and a suitable powder base such as lactose or starch. The powder composition may be provided, for example, in a capsule or drug package, or in a unit dosage form in gelatin or a blister pack, where the powder can be administered, for example, by an inhaler or blower.

If necessary, the above-mentioned preparation adapted to the sustained release of the active ingredient can be used.

The pharmaceutical compositions described herein may also contain other active ingredients such as antibacterial or preservatives.

Furthermore, it is contemplated that the therapeutic proteins disclosed herein can be used in combination with other therapeutic agents. For example, the present invention provides a method of pretreatment with a pharmaceutically effective amount of an antihistamine to minimize or prevent a potential injection-related anaphylactic reaction. For example, the antihistamine can be any pharmaceutically acceptable antihistamine (eg, diphenhydramine) disclosed herein and known in the art. In one embodiment, the antihistamine is administered at a dose between about 1 mg and about 10 mg / kg body weight. For example, antihistamines can be administered at a dose of about 5 mg / kg body weight. In one embodiment, a portable system connected to a vascular access port is used to apply the antihistamine for about 10 to about 90 minutes, for example about 30 to about 60 minutes, prior to administration of lysosomal acid lipase. Administer. In one embodiment, administration of diphenhydramine effectively suppresses a potential anaphylactic injectable response.

Antibodies to immunosuppressive agents such as antihistamines, corticosteroids, cilolimus, bocrosporin, cyclosporine, methotrexate, IL-2 receptors, before, during or after LAL administration, if the patient experiences an anaphylactic or adverse immune response. Antibodies to T cell receptors, antibodies to TNF-α or fusion proteins (infliximab, etanercept or adalimumab), CTLA4-Ig (eg abatacept), anti-OX-40 antibodies and the like can also be administered.

The present invention also contemplates therapeutic methods involving the administration of LAL-containing compositions in combination with one or more cholesterol lowering agents (eg, HMG-CoA reductase inhibitors). Non-limiting examples of such agents include atorvastatin (Lipitor® and Torvast®), fluvastatin (Lescol®), lovastatin (Mevacor®, Altocor®). ), Altoprev®), pitavastatin (Livalo®, Pitava®), pravastatin (Pravachol®, Selektine®, Lipostat®), Lovastatin (Crestor®) Trademarks)) and simvastatin (Zocor®, Lipex®).

Various conditions can be treated with the compositions or proteins described herein. For example, there is a condition in which the treatment method is known to a doctor who is skilled in the art. The present invention contemplates that such a condition can be treated using a Therapeutic protein (eg, LAL) produced in the avian system and containing a poultry-derived glycosylation pattern. That is, it is also contemplated to treat a condition known to be treatable with a therapeutic protein produced by conventional methods with a therapeutic protein produced as described herein. For example, with LAL produced as described herein, conditions caused by or associated with LAL deficiency or deficiency (collectively referred to as "LAL deficiency"), such as Wolman's disease and It can treat cholesteryl ester accumulation disease (CESD) and the like. In the LAL deficiency described herein, the expression of LAL is also reduced by a decrease or deficiency of LAL produced in the body (for example, gene mutation), or by a physiological factor or an environmental factor. Will be considered. Using LAL produced as described herein, other conditions such as atherosclerosis, fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis (NASH) and cirrhosis Etc. can also be treated. US Pat. No. 6,849,257 issued February 1, 2005, using LAL produced as described herein (this disclosure is incorporated herein by reference in its entirety). US Patent Application Publication No. 2009/0297496 published on December 3, 2009; US Patent Application Publication No. 2004/0223960 published on November 11, 2004; Published on November 15, 2009 Others, such as those disclosed in US Patent Application Publication No. 2007/0264249 (these disclosures (ie, each of the above four patent publications) are incorporated herein by reference in their entirety). The condition can also be treated.

LALs produced as disclosed herein can be used to treat certain conditions, including pancreatitis, such as chronic pancreatitis and / or acute pancreatitis, and alcohol-induced pancreatic damage such as alcoholic pancreatitis. Is also planned.

Using LALs made by any useful method, such as the methods disclosed herein, body tissues, such as, but not limited to, liver, spleen, gastrointestinal tract and cardiovascular tissue, etc. It is intended to treat diseases caused by alcohol-induced cell damage, including alcohol-induced cell damage that causes the accumulation of lipid esters. The present invention also contemplates treatment of malabsorption by LAL administration.

One aspect of the invention relates to a method of treating a patient, comprising administering to the patient a therapeutically effective amount of a composition comprising the recombinant human LAL described herein. The patient suffers from or can suffer from any condition, including those associated with LAL deficiency. In one embodiment, a therapeutically effective amount is an amount that increases the patient's red blood cell count by a desired amount. It is contemplated that LALs produced according to the present invention can be used, for example, to treat chronic kidney disease in which tissues are unable to sustain the production of lysosomal acid lipase.

It is also contemplated that LALs made by any effective method may be useful in the treatment of patients with Tangier disease and familial hypoalphalipoproteinemia. Tanzier's disease / familial hypoalphalipoproteinemia is associated with the accumulation of cholesterol esters in macrophages and, along with hepatic splenic enlargement and / or lymph node swelling, is a high-density lipoprotein (HDL) that can be treated with LAL. Accompanied by a decrease in. For example, it is not desired that the present invention be limited to a particular theory of action or mechanism of action, but impaired LAL activity reduces ABCA1 expression and, conversely, Tanzier's disease / familial hypoalphalipoproteinemia. It is considered that the increase in LAL activity obtained by administration of LAL to the patients in the above increases ABCA1 expression, and the influence of the ABCA1 gene whose functional activity is decreased due to polymorphism is overcome.

In general, in the treatment of a condition, the dose may vary depending on known factors such as the age, health condition, weight of the recipient, the type of treatment used in combination, the frequency of treatment, and the like. Generally, the dose of active ingredient can be between about 0.0001 and about 10 milligrams per kg of body weight. The exact dose, frequency of administration and duration of treatment can be determined by a skilled physician in the field of each therapeutic protein administration.

Furthermore, it has been found that 1 mg / kg or less may be effective in the treatment of LAL deficiency. The present invention presents a therapeutically effective amount of lysosomal acid lipase between once every 5 days to once every 25 days, eg, once every 7 days to once every 14 days, in mammals (eg, patients, preferably). Provides a cure for the condition, including administration to human patients). In one embodiment, the dose of lysosomal acid lipase administered may be between about 0.1 mg and about 50 mg / kg body weight, for example, the dose may be between about 1 mg and 5 mg / kg body weight.

In one particularly useful embodiment, the invention follows a dose of about 0.1 mg to 1.0 mg of lysosomal acid per kg of body weight, according to any therapeutically effective dosing regimen, such as the dosing regimen described herein. Provided is a cure for the condition by administering lipase.

The present invention provides a method of treating any complications of LAL deficiency in which administration of a therapeutically effective amount of LAL may be useful. In one embodiment, malabsorption and growth deficiency can be treated by the methods described herein. In another embodiment, the methods provided herein can be used to treat complications found in patients with LAL deficiency, including but not limited to hepatomegaly and liver damage.

The present invention provides treatment with any effective protein expression system as understood in the art, eg, recombinant LAL (eg, recombinant human LAL) that can be produced by transgenic mammals and transgenic birds. provide. Other protein expression systems may include, but are not limited to, cell culture systems, bacterial systems and plant systems.

The present invention comprises administering recombinant LAL as part of a pharmaceutically acceptable composition by any route that can achieve the intended therapeutic effect as determined by a skilled physician in the art. .. For example, a portable infusion pump connected to a vascular access port (eg, an indwelling port) can facilitate injection.

The invention also includes monitoring the clinical and pathological onset of conditions in mammals (eg, human patients), such as Wolman's disease and CESD. In one embodiment, the assessment is not particularly limited, but is limited to lipid analysis, chest X-ray, liver function tests, stool chart, plasma mevalonic acid, immunogenicity, plasma lysosome acid lipase, chitotriosidase, PARC, portal. It consists of portal hypertension, anthropometry, eg liver, pancreas and gastrointestinal volume and characterization using imaging techniques. For example, the imaging technique may consist of ultrasound, magnetic resonance imaging and nuclear magnetic resonance spectroscopy.

<p num="0147"> The present invention will be further illustrated by the following examples. The examples are for illustration purposes only and are not intended to limit the invention in any way and should not be construed as limiting.</p><p num="0148">Example 1 Construction of a vector (pALVIN-OVR1-I-hLAL-dSA) carrying a recombinant human lysosomal acid lipase (rhLAL) coding sequence The nucleotide sequence of the hLAL gene in the pALVIN-OVR1-I-hLAL-dSA vector encodes the same protein as the amino acid sequence of the protein produced by the human lysosomal acid lipase gene (GenBank accession number: NP_000226) (Fig. 1). ). Transcription of this sequence followed by translation of the resulting mRNA produced a 399 amino acid precursor protein, which was processed and described in SEQ ID NO: 1 for human LAL (GenBank accession number: NP_000226) (Fig. 1). ) Becomes the same mature 378 amino acid protein. Expression of the hLAL gene of this example (see Figure 2 for cDNA sequence) is regulated by non-coding elements derived from the ovoalbumin gene, including enhancer sequences, promoter sequences, intron sequences, and 5'and 3'untranslated sequences. To. The ovalbumin gene produces ovalbumin, the major protein component of egg white. The activity of the chicken ovalbumin promoter is extremely specific for cells in the oviduct of chickens that produce egg white, and its expression in other tissues is extremely low.</p><p num="0149"> A replication-deficient retro that stably integrates the hLAL transgene into the genome of the wound initiator (XLL109) using the plasmid vector pALVIN-0VR1-I-hLAL-dSA (Fig. 3A; nucleotide sequence shown in Fig. 4). A virus (RDR) was generated. This plasmid vector contains the retroviral nucleotide sequences required for viral RNA packaging, reverse transcription and integration, but does not contain intact sequences for the viral gag, pol and env genes. The methods used to make retroviral vectors and their use in subsequent gene transfer procedures are described herein.</p><p num="0150"> The retroviral portion of pALVIN-OVR1-I-hLAL-dSA is based on the ALV vector pNLB. The pNLB was modified so that the LTR was self-inactive (SIN) (Fig. 3B). To achieve this, the 273 bp 3'LTR containing the U3 region enhancer and CAAT box was removed. The 5'LTR is also normally inactivated because the 3'end inactivated U3 region of the retrovirus sequence acts as a template for the new U3 region present at the 5'end of the integrated provirus. .. Deletion of the LTR sequence in the SIN construct reduces promoter interference from the LTR with respect to the internal promoter and minimizes the likelihood of replication retrovirus formation by sequence recombination. The new vector is named pALVIN after the ALV inactivating vector.</p><p num="0151"> Downstream of the 5'LTR is the partial gag and env coding sequences inherited from the pNLB vector. A small portion (12%) of the gag protein precursor sequence remains in pALVIN-OVR1-I-hLAL-dSA (55% of the p19 mature peptide sequence), and a small portion of the RAV2 env precursor sequence (55%). 1.7%) remains (GenBank accession number: AF033808). These truncated gag and env regions are unable to produce the functional proteins required to give rise to replicative retroviruses (Cosset, 1991).</p><p num="0152"> The transcriptional and translational regulatory elements of the chicken ovalbumin gene were inserted into pALVIN to generate pALVIN-OV-1.1-I (sequence is shown in Figure 6; SEQ ID NO: 8). The first part of pALVIN-OV-1.1-I consists of a 1.1 kb proximal promoter region, a contiguous part of the chicken ovalbumin gene containing parts of the first exon, first intron and second exon. There is. The next part is a stuffer-inserted fragment that replaces the ovalbumin protein coding sequence. The stuffer is followed by the 3'untranslated region (UTR) of the chicken ovalbumin gene, which contains a sequence that facilitates proper processing of mRNA, including polyadenylation. Generally, the stuffer fragment is replaced with a DNA fragment that encodes the desired protein, in this case hLAL. As a result, it has specific elements that promote regulated transcriptional expression and translation of mRNA in the transgenic chicken oviduct, faithfully mimics the regulation of endogenous ovalbumin mRNA, and is highly advanced in the protein of interest in egg white. A vector that allows expression is obtained.</p><p num="0153"> The pALVIN-OV-1.1-I vector contains the first intron of the ovalbumin gene. Since introns are susceptible to splicing during the generation and packaging of the retroviral RNA genome, we inserted the expression cassette in the opposite direction to the LTR. In this way, the intron becomes unrecognized within the retroviral RNA and is packaged unspliced. For convenience, all maps herein are drawn with the LTR in the opposite direction and the expression cassette in the forward or clockwise direction.</p><p num="0154"> Using pALVIN-OV-1.1-I as the basic vector, the hLAL coding sequence (CDS) was inserted into it. Two DNA fragments of the hLAL adapter and Syn hLAL that make up the sequences required for compatibility with hLAL CDS and pALVIN-OV-1.1-I were synthesized at Integrated DNA Technologies (Coralville, Iowa) (Fig. 7). See and 8; SEQ ID NOs: 9 and 10). Replacing the stuffer region with hLAL CDS by inserting the 229 bp Hpal / BamHI fragment of the hLAL adapter and the 1113 bp BamHI / BstBI fragment of Syn hLAL into the 7882 Hpal / BstBI fragment of pALVIN-OV-1.1-I, pALVIN-OV -1.1-I-hLAL was prepared.</p><p num="0155"> It was revealed that there is a mysterious splice site in the antisense strand of hLAL CDS that interferes with the packaging of intact retroviral RNA. This mysterious splice site was removed by modifying the DNA sequence without altering the amino acid sequence of hLAL. This modification was carried out using the primer 5'-AGAAACTGAGAGTGTCTTAT-3'(SEQ ID NO: 12) and the primer 5'-TGACAGCTGTGGATCCAGAAACAAACATG-3' (SEQ ID NO: 13) in the polymerase chain of region 232 to 534 of pALVIN-OV-1.1-I-hLAL. Amplification was performed to produce a 329 bp amplicon. This amplicon was digested with BamHI and SexAI and ligated into an 8940 bp BamHI / SexAI fragment of pALVIN-OV-1.1-I-hLAL to generate pALVIN-OV-1.1-I-hLAL-dSA.</p><p num="0156"> A putative promoter enhancer containing the DNase hypersensitive site III (DHSIII) of the chicken ovalbumin gene (positions -3819 to -2169 from the OV promoter initiation site) (Kaye, Bellard et al., 1984) was pALVIN-0V-1.1-I- It was inserted into hLAL-dSA to prepare pALVIN-OVR1-I-hLAL-dSA. This was done as follows. A DNA fragment containing a DHSIII enhancer and a proximal OV promoter called the 1.1 kb OVR1 promoter (see Figure 9; sequence number 11) was isolated by digestion with Xho I and Blp I. Regions 6752-7974 of pALVIN-OV-1.1-I using primers 5'-GCCGCTCGAGCGAGGAATATAAAAAAATT-3'(SEQ ID NO: 14) and 5'-TCCGCGCACATTTCCCCGAA-3' (SEQ ID NO: 15) to facilitate subcloning. PCR amplification was then performed, followed by digestion with NgoMI and XhoI to generate PCR for the adapter fragment pSIN-OV-1.1-I. Insert the 2772 bp XhoI / BlpI fragment of the OVR1 promoter and the 1067 bp NgoMI / XhoI fragment of the pSIN-OV-1.1-I PCR into the 7043 bp NgoMI / BlpI fragment of the pALVIN-OV-1.1-I-hLAL-dSA. A pALVIN-0VR1-I-hLAL-dSA was made (see Figure 10 for a schematic diagram of the construction of the pALVIN-0VR1-I-hLAL-dSA). Construction of a retroviral vector segment of a vector called pALVIN (also known as pAVIJCR-A395.22.3.1-KM or pALV-SIN) is described in US Patent Application No. 2008/0064862.</p><p num="0157"> In addition, the promoters and / or vectors disclosed in U.S. Patent Application Publication No. 2008/0064862, published March 13, 2008, the entire contents of which are incorporated herein by reference. The production of LAL according to the present invention used is included.</p><p num="0158">Example 2 Virus particle production Using the retrovirus gene transfer method as follows, a transgenic male XLL109 carrying the hLAL transgene in the genome, which is a G0 wound initiator, was prepared. Transient transfection of an immortalized chicken fibroblast line generated replication-deficient virus particles carrying the pALVIN-OVR1-I-hLAL-dSA vector. These chicken fibroblasts were simultaneously transfected with three plasmids, pALVIN-OVR1-I-hLAL-dSA, pCMV-gag-pol and pCMV-VSV-G. pCMV-gag-pol expresses the gag and pol genes of the avian leukemia virus RAVI strain. pCMV-VSV-G expresses the enveloped protein of the vesicular stomatitis virus. Four hours after transfection, the medium was replaced with DMEM supplemented with 10% fetal bovine serum, 100 units / mL penicillin and 100 μg / mL streptomycin. Medium was collected 48 hours after transfection, filtered through a 0.45 micron filter (Millipore) and concentrated by ultracentrifugation. Retroviruses carrying the concentrated ALVIN-OVR1-I-hLAL-dSA transgene were collected and used for early embryonic transduction. Since "p" is a symbol of a plasmid-type vector, once the transgene is in the form of a packaged vector or integrated transgene, the name of the transgene will not have "p". Please note.</p><p num="0159">Example 3 Gene transfer into the embryo Integration of the ALVIN-OVR1-I-hLAL-dSA expression cassette into the embryonic genome was performed by early embryonic transduction (Speksnijder and Ivarie, 2000). Fertilized eggs of freshly laid white Leghorn were obtained from a breeding colony. A hole was made in the shell so that it could come into contact with the embryo. 7 microliters of concentrated replication-deficient retrovirus particles carrying the ALVIN-OVR1-I-hLAL-dSA expression cassette were injected into the embryonic subembryonic cavity. Eggs were sealed with thermal adhesive and then incubated under standard conditions for hatching. Individual identification markers were attached to the offspring produced by the above injections at hatching for identification and tracking. When the hLAL transgenes were analyzed by real-time PCR using PCR primers specific for the hLAL coding sequence in the blood samples of the offspring, these were positive for the transgenes (described below). This showed that the gene transfer procedure was successful. For real-time PCR assays for hLAL transgenes, Taqman® Chemistry (Applied) Biosystems) is used. The forward and reverse primers were 5'-ACGACTGGCTTGCAGATGTCT-3'(SEQ ID NO: 16) and 5'-CCCCAAATGAAGTCAAGATGCT-3' (SEQ ID NO: 17), respectively. The Taqman® probe sequence was 5'-CCGGAATGCTCTCATGGAACACCAA-3'(SEQ ID NO: 18), labeled with FAM at the 5'end (as an emitter) and Iowa Black at the 3'end (as a quencher). .. 30 μl of primer, probe and 1 μl of extracted DNA from Taqman® Universal Master Mix (Applied) Added to Biosystems). Control reactions included various dilutions of plasmids with hLAL sequences and wild-type chicken-derived DNA (data not shown). Standard cycling parameters were used for the Applied Biosystems 7500 Fast Real-Time PCR System.</p><p num="0160">Example 4 Identification of G0 wound initiator Semen was collected from sexually mature males and DNA was extracted and assayed using the hLAL real-time PCR assay. Transgene copy numbers in each sample were estimated using a known reference material (plasmid with the hLAL gene) mixed with negative control semen DNA. Estimates by real-time PCR showed that the transgene cassette DNA content of male XLL109 was at a level capable of transmitting the transgene to its offspring. This XLL109 male was used as a G0 transgenic wound initiator and crossed with a non-transgenic chicken to give birth to a G1 hemizygous transgenic chicken.</p><p num="0161">Example 5 Breeding and characterization of hemizygous G1 hectria Progeny born to the transgenic wound initiator XLL109 were tested for the presence or absence of transgene in blood cell DNA using the hLAL real-time PCR assay. Blood was drawn from offspring 1-2 weeks of age and DNA was extracted using high-throughput techniques (Harvey et al., 2002). DNA solutions were not quantified prior to the Taqman assay to facilitate high-throughput screening. Typically, 1 μl of DNA solution contains 50-400 ng of DNA sufficient to produce a positive amplification signal. A total of 1,322 chickens parented by XLL109 were tested, and blood was collected again from positive offspring for confirmation. PCR results showed that 22 offspring were positive for the ALVIN-OVR1-I-hLAL-dSA transgene. An example of Taqman's results is shown in Figure 11.</p><p num="0162">Example 6 Identification and characterization of highly expressed strains One G1 chicken, 1LL7466, laid eggs in the egg white that contained significantly higher levels of rhLAL protein than other G1 chickens. Southern blot analysis was performed on 1LL7466 and sibling G1 males to identify sibling males with the same integration sites as high-expressing chickens. Digestion was performed with a restriction enzyme (BlpI) that cleaves only once in the transgene, and Southern blots were probed with a segment of the ovalbumin promoter or the hLAL coding sequence (Figs. 12A-12D). The location of the second restriction site is within the adjacent genomic region and depends on the site of integration. Therefore, the size of the BlpI band detected by the OV or hLAL probe is unique to each strain that arises.</p><p num="0163"> The OV probe detected a 4.1 kb single band in BlpI digested DNA from wild-type chickens, which corresponded to the predicted size of the BlpI segment of the endogenous ovalbumin gene in the chicken genome (Fig. 12B and). 12D). A second band of 4.3 kb was detected in chicken 1LL7466, which corresponded to the transgene band. Three additional female siblings 1LL10409, 1LL10686 and 1LL12058, and three additional male siblings 1LL8922, 1LL9330 and ILL11217 showed a 4.3 kb band, which means that these siblings can be of the same strain. (Figs. 12B and 12D).</p><p num="0164"> As expected, wild chickens because the DNA sequence of the chicken lysosomal acid lipase gene and the coding sequence of the recombinant human lysosomal acid lipase are sufficiently differentiated to prevent hybridization under the conditions used in the Southern assay above. No bands were detected by the hLAL probe in the derived DNA (Fig. 12C). In the BlpI digested genomic DNA from the same chicken that was positive for the 4.3 kb band detected by the OV probe, the hLAL probe detected a single band of approximately 10.6 kb, which is the result of these seven G1 chickens. Show that they have the same integration site and therefore the same strain.</p><p num="0165"> No other bands were detected, indicating that 1LL7466, 1LL10409, 1LL10686, 1LL12058, 1LL8922, 1LL9330 and 1LL11217 all have a single integration site.</p><p num="0166"> Southern analysis also showed that the transgene was integrated because the bands detected by the OV and hLAL probes differed in size and were larger in size than the bands derived from a single transgene. A map showing the expected structure of the integrated transgene and the location of the BlpI site within the adjacent genomic region is shown in Figure 12A.</p><p num="0167"> We confirmed that the transgene is intact in two steps. First, the hLAL coding sequence was isolated from 1LL7466 by PCR. Both strands of the PCR product were sequenced from the hLAL start codon to the stop codon. The DNA sequence was quite as expected, indicating that the DNA sequence of the coding region within the transgene was not altered at all. Next, Southern blot analysis was performed using the restriction enzyme ApaLI, which digests the intact transgene into two segments, 3.6 kb and 3.8 kb (Fig. 13A). Both 3.6 kb and 3.8 kb bands were detected in the G1-derived ApaLI digested genomic DNA, indicating that the transgene was fully integrated (Fig. 13B).</p><p num="0168">Example 7 Breeding and characterization of G2 The lineage of hLALG2 from a single G0 wound initiator XLL109 is shown in Figure 14. At the G1 stage, transgenes were characterized for copy number, consistency, hLAL sequence and integration site, and 7 G1 transgenics were identified and characterized (4 chickens and 3 male chickens). .. Non-transgenic chickens were artificially inseminated with semen collected from G1 male parents 1LL8922, 1LL9330 and 1LL11217 to breed G2 (Fig. 14). Each fertilized chicken, its egg and the next offspring were bred in isolation from other offspring. Hatched offspring were tested for the presence or absence of the hLAL transgene using the hLAL real-time PCR assay. Since the G1 wound initiator is hemizygous with respect to the transgene, it was expected that half of the offspring would be transgenic G2. Of the 610 G2 offspring analyzed so far, 330 or 54% were transgenic.</p><p num="0169">Example 8 Gene analysis of hLAL birds After each G2 chicken was identified by the hLAL real-time PCR assay of blood DNA, the following gene assay was performed on the production line: the hLAL gene was PCR amplified and sequenced from the blood DNA to 100% of the human sequence. The identity was confirmed; the transfer gene integration site was confirmed by integration site PCR as described above. PCR sequencing and integration site analysis were performed on the following chickens: 10 chickens from less than 10 chicken-producing strains; 10% of chickens from 11-100 chicken-producing strains (minimum 10 chickens); 101-1000 5% chickens from feather chicken-producing strains (minimum 10); 1% chickens from 1001 to 10,000 chicken-producing strains (minimum 50); 0.1% chickens from chicken-producing strains over 10,001 (minimum 100 chickens) ). Detailed recording was continued for each stage of growth and production.</p><p num="0170">Example 9 Purification of hLAL from egg white Egg white (EW) containing LAL was solubilized at pH 6 overnight and clarified by centrifugation (or deep filtration) using 0.2 μm filtration. EW was adjusted to pH 6 with 1M NaO Ac buffer (pH 4).</p><p num="0171"> The clarified EW was loaded onto a phenyl-HIC column (EW: column size = 2: 1) equilibrated with 20 mM phosphate / 137 mM NaCl buffer (pH 6). After loading was complete, the column was washed with equilibration buffer and 5 mM phosphate buffer (pH 6). LAL was eluted with 30% propylene glycol containing 5 mM Tris buffer (pH 7.2).</p><p num="0172"> The eluted LAL fraction was adjusted to pH 5 with 1 M acid and then loaded onto a GigaCap S column (EW: column size = 10: 1). The column was equilibrated with 50 mM NaO Ac buffer (pH 5). After the loading was complete, the column was washed with equilibration buffer. LAL was eluted with 50 mM NaOAc / 60 mM NaCl (pH 5).</p><p num="0173"> The LAL fraction from the GigaCap S column was adjusted to pH 6 with 1 M Tris buffer and then loaded onto a butyl-HIC column (EW: column size = 10: 1). The column was equilibrated with 20 mM phosphate / 137 mM NaCl buffer (pH 6). After loading was complete, the column was washed with equilibration buffer and 5 mM phosphate buffer (pH 6). Pure LAL was eluted with 50% propylene glycol containing 5 mM Tris buffer (pH 7.2). The purification step of hLAL from egg white is illustrated in FIG.</p><p num="0174">Example 10 Carbohydrate analysis of transgenic bird-derived hLAL The oligosaccharide structure of avian-derived human LAL was determined using the following analytical techniques known to those skilled in the art.</p><p num="0175"> 200 micrograms, 1 mM CaCl₂Digested with trypsin and chymotrypsin at 37 ° C for 18 hours in 0.1 M tris-HCl (pH 8.2) containing. Digestive products were concentrated and contaminants were removed by C18 cartridge column. After concentration, the glycopeptide was digested in 50 μl of 20 mM sodium phosphate buffer (pH 7.5) at 37 ° C. for 18 hours with 2 μl of PNGase F (7.5 units / ml). The dissociated oligosaccharides were passed through a Sep-Pak C18 cartridge column to separate them from peptides and enzymes.</p><p num="0176"> The glycan fraction was dissolved in dimethyl sulfoxide and then permethylated by the methods of Anumula and Taylor (Anumula and Taylor, 1992). Water was added to stop the reaction and per-O-methylated carbohydrates were extracted with dichloromethane. Per-O-methylated glycans were dried under a stream of nitrogen.</p><p num="0177"> Reflector using MALDI / TOF-MS (matrix-assisted laser desorption / ionization time-of-flight mass spectrometry) as a matrix using α-dihydroxybenzoic acid (DHBA, 50% methanol: 20 mg / mL solution in water). It was done in cation mode. The entire spectrum was obtained using Microflex LRF (Bruker).</p><p num="0178"> After dissociation and purification from the peptide backbone, MALDI-TOF-MS analysis and ESI MS / MS (electrospray ionization tandem mass spectrometry) were performed on oligosaccharides as understood in the art. Samples of individual polysaccharide species were also digested with specific enzymes and the digested products were analyzed by HPLC as understood in the art.</p><p num="0179"> It is believed that there are approximately 6 N-linked glycosylation sites on human LAL. See Zschenker et al. (2005) J. Biochem., Vol 137, p387-394 (this disclosure is incorporated herein by reference in its entirety). This reference also suggests that O-linked glycosylation sites may be present on human LALs. The identified N-linked oligosaccharide structure is shown in FIG.</p><p num="0180"> The data revealed that many or all of these structures are found as N-linked glycosylated structures in LALs produced according to the present invention (Fig. 16). For example, it is found that An is bound to LAL produced according to the present invention. For example, it is found that On is bound to LAL produced according to the present invention. For example, it is found that at least one of Bn, Cn and Dn is bound to LAL produced according to the present invention. For example, it is found that at least one of En and Fn is bound to LAL produced according to the present invention. For example, it is found that at least one of In and Jn is bound to LAL produced according to the present invention. For example, it is found that at least one of Kn and Ln is bound to LAL produced according to the present invention. For example, it is found that at least one of Mn and Nn is bound to LAL produced according to the present invention. For example, it is found that Gn is bound to LAL produced according to the present invention. For example, it is found that Hn is bound to LAL produced according to the present invention.</p><p num="0181">Example 11 Transgenic bird-derived LAL N-glycan species A purified sample (600 μg / sample) of transgenic bird-derived hLAL was subjected to ultrapure water (nanopure water) at 4 ° C for about 24 hours using a Tube-O-Dialyzer (4.0 kDa cut-off membrane; G Biosciences). Dialysis was performed to remove salts and other contaminants. Ultrapure water was changed 4 times during the total dialysis time.</p><p num="0182"> After dialysis, each sample is placed in 3 aliquots, ie about 1/4 of the sample weight for neutral and amino sugar analysis and about 1/4 of the sample weight for mannose-6-phosphate analysis. About 1/2 of was separated for oligosaccharide profiling. Hydrolyzed aliquots for neutral and amino sugar analysis at 100 ° C for 4 hours with 2N trifluoroacetic acid (TFA) and aliquots for mannose-6-phosphate analysis at 100 ° C for 1.5 hours, 6.75N TFA Hydrolyzed by. Then N hydrolyzate₂Dry under, 50 μL H₂It was thawed again to 0, sonicated in ice for 7 minutes and transferred to an infusion vial. However, the neutral and amino sugar samples were diluted twice because the peaks obtained from the original lysed hydrolyzate were too large.</p><p num="0183"> A mixture of known molar numbers of standards for neutral and amino sugars and standards for mannose-6-phosphate was hydrolyzed in the same manner as the sample for the same amount of time. Mixtures of neutral and amino sugar standards at four concentrations (0.2, 0.4, 0.8 and 1.6 nanomoles of Fuc and GalNAc per 10 μl; 0.5, 1.0, 2.0 and 4.0 nanomoles of GlcNAc per 10 μl; 0.3, 0.6, 1.2 and 10 μl. 2.4 nanomoles of Gal and Man; as well as 0.1, 0.2, 0.4 and 0.8 nanomoles of Glc per 10 μl) and mannose-6-phosphate (640, 1280, 2560, 5120 picomoles per 0 μl) were prepared and calibration equations were established. The number of moles of each sugar in the sample was quantified from the calibration equation by linear interpolation.</p><p num="0184"> Neutral and amino sugars as well as mannose-6-phosphate were analyzed by HPAEC using a Dionex ICS3000 system equipped with a gradient pump, an electrochemical detector and an autosampler. Individual neutral and amino sugars as well as mannose-6-phosphate were separated by a Dionex CarboPac PA20 (3 × 150 mm) analytical column with an amino trap. The gradient program includes eluent A (degassed nanopure water) and eluent B (200 mM NaOH) for neutral and amino sugars, eluent C (100 mM NaOH) and eluent D (100 mM NaOH) for mannose-6-phosphate. 1M sodium acetate in NaOH) was used. Injections (10 μL / injection) were performed every 40 minutes for neutral and amino sugar measurements and every 35 minutes for mannose-6-phosphate measurements. All methods are Hardy and Townsend (Hardy, MR and Townsend, RR, High-pH anion-exchange chromatography of glycoprotein-derived Based on the protocol described by carbohydrates , 1994, Methods Enzymol. 230: 208-225). Instrument control and data acquisition were performed using Dionex chromeleon software. Results are shown in Table 1 below. The sample is ovomucoid purified from EW.</p><p num="0185"><tables num="1"><img id="000006" he="136" wi="140" file="JP2016190867A_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0186">Structural features of LAL LAL has six potential sites for N-linked glycosylation in its amino acid sequence, namely Asn.³⁶, Asn⁷², Asn¹⁰¹, Asn¹⁶¹, Asn²⁷³And Asn³²¹Have. Five of these, namely Asn³⁶, Asn¹⁰¹, Asn¹⁶¹, Asn²⁷³And Asn³²¹Is glycosylated, whereas Asn⁷²Is non-glycosylated or substantially non-glycosylated (substantially non-glycosylated is Asn in a mixture of LAL molecules.³⁶, Asn¹⁰¹, Asn¹⁶¹, Asn²⁷³And Asn³²¹Less than any of Asn⁷²Means that is glycosylated). Therefore, one aspect of the present invention is Asn.⁷²LALs that are non-glycosylated and / or substantially non-glycosylated in (eg, human LALs), as well as the production and use of such LALs. However, glycosylated Asn⁷²LAL having is within the scope of the present invention. The N-glycan structure is mainly composed of a mixture of bi-branched, tri-branched and quaternary structures having N-acetylglucosamine, mannose and mannose-6-phosphate (M6P) as major sugars. Each site appears to have an advantageous set of structures (Table 2 and FIG. 17) that is an aspect of the invention. For example, M6P modified N-glycans are Asn¹⁰¹, Asn¹⁶¹And Asn²⁷³Exists in. The non-phosphorylated structure is typical of N-glycans found in endogenous egg white proteins. No O-linked glycans were detected, as determined by the absence of N-acetylgalactosamine (GalNac). No sialic acid was detected, which is consistent with the already determined N-glycan structure of other endogenous and exogenous proteins produced according to the present invention. The present invention includes one or more LALs glycosylated with the oligosaccharide structures disclosed herein.</p><p num="0187"><tables num="2"><img id="000007" he="154" wi="141" file="JP2016190867A_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0188">Method The monosaccharide composition, including neutral sugars, amino sugars and M6P, was determined qualitatively and quantitatively using high pH anion exchange chromatography-pulse amperometric detection (HPAEC-PAD).</p><p num="0189"> Data from several mass spectrometers (MALDI-TOF, NSI-MS / MS and glycopeptide LC-MS) were used to determine the predominant glycan structure.</p><p num="0190"> MALDI-TOF was effective in determining neutral N-glycans and was able to detect phosphorylated N-glycans (Fig. 18). NSI-MS / MS was used to determine the properties of micropeaks in the MALDI-TOF spectrum, some of which were due to phosphorylated N-glycans (Fig. 19). Efforts to improve the ability of MALDI-TOF to detect phosphorylated N-glycans have not been fruitful.</p><p num="0191"> LC / MS of glycopeptides was able to determine the neutral and phosphorylated structures and the location of specific structures in the amino acid sequence of LAL (data in Figures 17 and 2). To summarize).</p><p num="0192"> LAL was treated with phosphatase and analyzed to determine which peaks in the HPAEC-PAD chromatogram were due to phosphorylated N-glycans (Fig. 3). The area under the curve (AUC) decreased for the peaks of groups C and D, while the peaks of group A became more prominent. The group B peak was unchanged compared to the other peaks. Based on the knowledge that retention time is proportional to the degree of charge (due to phosphorylation or sialylation), group C is composed of N-glycans (mono M6P) with one phosphate and group D is two phosphorus. It is considered to be composed of N-glycan (bis-M6P) having acid.</p><p num="0193"> Retention time was also influenced by the composition and relative structural position of the neutral and amino monosaccharides. Examples of such include the presence of galactose, the presence of bifurcated GlcNac and the degree of GlcNac substitution. Such factors contribute to the diversity of peaks in the HPA EC-PAD chromatogram.</p><p num="0194">Example 12 In vitro enzyme activity analysis of transgenic bird-derived hLAL in egg white The activity of lysosomal acid lipase in egg white is basically described by Yan et al. (2006), American Journal of Pathology, Vol.169, No.3, p916-926. Fluorescent substrate 4-methylumbelliferyl oleate assay was used as described in.</p><p num="0195"> A stock solution of 4-methylumbelliferyl oleate (4-MUO) consisting of 2.5 mM 4-MUO in 4% Triton X-100 was prepared. Assays were performed on microtiter plates in which each well contained 0.2 M sodium citrate (pH 5.5) 62.5 μl, 12.5 μl egg white sample and 25 μl 2.5 mM 4-MUO in 0.01% Tween 80. Fluorescence changes were monitored at 37 ° C for 30 minutes using a Bio-Tek Synergy HT fluorescence measurement microplate reader (excitation 360 nm and emission 460 nm). Prior to the assay, egg whites containing hLAL were diluted to enzyme concentrations that produced a reaction that lasted linearly for at least 30 minutes. The reaction was stopped with 50 μl of 0.75 M Tris HCl (pH 8.0) and endpoint fluorescence signals were measured with the same plate readers used above (excitation 360 nm and emission 460 nm).</p><p num="0196"> The active unit was determined using 4-methylumbelliferyl as a standard. One unit (U) is defined as the amount of enzyme that produces 1 μmol of 4-methylumbelliferyl per minute under the above assay conditions. Egg white without hLAL was used as a negative control.</p><p num="0197"> Egg white samples positive for hLAL had an activity between 1 U and 100 U per mL of egg white. The egg whites of 21 G1 chickens were analyzed. Egg whites of 10 chickens were positive in the hLAL activity test.</p><p num="0198">Example 13 In vitro analysis of transgenic bird-derived LAL The ability of LAL produced within transgenic avian oviduct cells (referred to herein as "SBC-102", "bird-derived LAL", "LAL" or "hLAL") to bind to cells and into the lysosomal compartment The ability to migrate was examined in vitro using macrophages and fibroblasts. Fluorescently labeled SBC-102 was localized to lysosomes when incubated with macrophage cells. This effect could be attenuated using a mannose polysaccharide competitor, suggesting uptake by the N-acetylglucosamine / mannose (GlcNAc / mannose) receptor and their cells as a cognitive mechanism. .. SBC-102 increased cell-bound LAL activity in LAL-deficient human fibroblasts and normal mouse fibroblasts after incubation in vitro, which means that exposure to SBC-102 resulted in deficient enzyme activity. It shows that a substantial replacement of can occur.</p><p num="0199"> Mannose-6-phosphate (M6P) is present in the oligosaccharide structure of SBC-102, which has been shown to be involved in lysosomal enzyme delivery to a wide variety of cell types via ubiquitous M6P receptors. ..</p><p num="0200"> LAL was purified from female hen transgenic egg whites. Oregon Green NHS was obtained from Invitrogen (# 0-10241). Rat alveolar macrophage line NR8383 and mouse fibroblast line NIH-3T3 were obtained from ATCC. LAL-deficient Wolman disease fibroblasts were obtained from the Coriell Institute for Medical Research and LysoTracker® Red was obtained from Invitrogen .</p><p num="0201">Enzyme Labeling: 4 mg transgenic bird-derived LAL in PBS was labeled with Oregon Green as recommended by the manufacturer, then the reaction was dialyzed against PBS and then concentrated.</p><p num="0202">Macrophage uptake: Fluorescently labeled transgenic bird-derived LAL (5 μg / mL) and LysoTracker® Red were incubated with NR8383 cells for 2 hours. Cells were examined by confocal fluorescence microscopy at 488 nm and then at 514 nm using continuous scan mode.</p><p num="0203">Competitive inhibition by mannan: Fluorescently labeled SBC-102 (5 μg / mL) and mannan were incubated with NR8383 cells for 2 hours. Cells were trypsinized and LAL uptake was measured by a fluorescence-marked cell preparative method using median fluorescence intensity as the endpoint.</p><p num="0204"> After the transgenic bird-derived LAL was taken up, the ability to be taken up into the lysosomes of the target cells was examined using the macrophage cell line NR8383. Fluorescently labeled transgenic bird-derived LAL and lysosomal marker "LysoTracker® Red" (Invitrogen ) were incubated with cells for 2 hours. Next, the co-localization of transgenic bird-derived LAL and lysosomal markers in the lysosomes of the cells was examined by confocal fluorescence microscopy using a continuous scan mode (Fig. 20). LAL showed localization to lysosomes, which is consistent with similar in vitro experiments with rhLAL from various sources.</p><p num="0205"> The binding specificity of transgenic tri-derived LAL for the GlcNAc / mannose receptor was evaluated by a competitive binding assay using the macrophage cell line NR8383 (Fig. 21). 5 μg / mL fluorescently labeled (Oregon Green) transgenic bird-derived LAL and various concentrations of mannose-containing oligosaccharide mannan were co-incubated with cells for 2 hours. The relative inhibition of transgenic bird-derived LAL uptake by mannan compared to a mannan-free control was quantified by fluorescence-marked cell preparative analysis using median fluorescence intensity as the endpoint. Mannose dose-dependent inhibition of transgenic bird-derived LAL binding / uptake was observed, consistent with transgenic bird-derived LAL: GlcNAcR interactions.</p><p num="0206"> In addition, competitive experiments with mannose-6-phosphate showed mannose-6-phosphate mediated uptake in fibroblasts (results not shown).</p><p num="0207"> The ability of exposure to transgenic bird-derived LAL to increase intracellular LAL activity was examined in vitro using both normal and LAL-deficient cells. Fibroblasts isolated from Wolman's disease patients and normal mouse fibroblasts (NIH-3T3) were incubated for 5 hours in the presence of transgenic bird-derived LAL at concentrations of 0, 0.16 or 0.5 μg / mL. Cells were then washed to remove non-specific signals and cell lysates were assayed for LAL activity using a 4-MUO substrate. Endogenous cell-bound LAL activity was lower in Wolman's disease fibroblasts compared to NIH-3T3, and a dose-dependent increase in activity was seen in both cell types after incubation with transgenic bird-derived LAL ( Figure 22).</p><p num="0208">Example 14 In vivo analysis of transgenic bird-derived LAL LAL-deficient Yoshida rats (ie, homozygous) (Kuriyama et al. (1990), Journal of Lipid Research, vol.31, p1605-1611; Nakagawa et al. (1995), Journal of Lipid Research, vol.36, p2212-2218; And Yoshida and Kuriyama (1990), Laboratory Animal Science, vol.40, p486-489), starting at 4 weeks of age, once a week for 4 weeks, SBC-102 (5 mg / kg, IV) or placebo. Treated with. At each dosing, SBC-102 was injected into the rat tail vein at two equal doses (2.5 mg / kg) every 30 minutes. Rats and same-age wild-type controls were examined 1 week after the last dose. The analysis was performed by triple iteration.</p><p num="0209"> Macroscopic pathological examination of SBC-102 treated individuals showed normalization of liver color in addition to reduced organ size. SBC-102 treated rats showed substantially normal liver tissue growth, in stark contrast to solvent treated rats, which had a significant accumulation of foamy macrophages. LAL^-/-Elevated blood alanine transferase and aspartate transferase levels in rats were also decreased in SBC-102 treated rats (not shown).</p><p num="0210"> The mass of internal organs and tissues of each rat was determined. The data is shown in FIG. Organ size is LAL^-/-Rat and LAL^+/+In rats, solvent or SBC-102 is administered at 5 mg / kg once weekly for 4 weeks and then expressed as a percentage of body weight determined at 8 weeks of age.</p><p num="0211"> As shown in FIG. 24, the body weight of SBC-102-treated or solvent-treated Yoshida rats was compared with that of wild-type rats. LAL^-/-Rats received SBC-102 (5 mg / kg) or solvent as a single or divided dose (administered within 4 hours) by IV injection. LAL^+/+Rats are littermates of the same age.</p><p num="0212">Example 15 Triglyceride analysis Triglyceride analysis was performed on the liver and spleen tissues of wild-type, homozygous, placebo-treated and homozygous SBC-102-treated rats. Triglyceride analysis was performed in triple iterations using standard methods (ie, MBL International's Triglyceride Quantification Kit, Catalog No. JM-K622-100).</p><p num="0213"><tables num="3"><img id="000008" he="32" wi="141" file="JP2016190867A_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0214">Liver substrate level Figure 25 shows 5 mg · kg of solvent or SBC-102 in WT and LAL deficient rats.^-1Shows liver cholesterol, cholesteryl ester and triglyceride levels determined at 8 weeks of age after administration once weekly for 4 weeks.</p><p num="0215">Example 16 Dose-response experiment Based on the study conducted above, the pharmacodynamic (PD) effect of the dose range and dosing regimen (weekly and biweekly) of LAL (SBC-102) was LAL.^-/-It was examined in rats. In these experiments, SBC-102 was administered by IV injection at 0.2, 1, 3 and 5 mg / kg biweekly or at 0.35, 1 and 5 mg / kg weekly doses starting at 4 weeks of age for 1 month. .. The results showed improved body weight (BW) gain (Fig. 26), organ hypertrophy (Fig. 27) and tissue substrate levels (Fig. 28). Blood transaminase levels also decreased with increasing SBC-102 dose, reaching virtually wild-type levels at high doses.</p><p num="0216">Example 17 Administration of recombinant LAL in rat model Effects of repeated doses of recombinant human lysosomal acid lipase (LAL) on body weight, tissue triglycerides and cholesterol, hepatomegaly, splenomegaly, lymphadenopathy, intestinal weight, and other parameters, Yoshida and Kuriyama (1990), Laboratory Animal LAL-deficient Donryu rats described in Science, vol.40, p486-489 (Kuriyama et al. (1990) Journal of Lipid Research, vol.31, p1605-1611; Nakagawa et al. (1995), Journal of Lipid Research, vol. See also 36, p2212-2218) (this disclosure is incorporated herein by reference in its entirety).</p><p num="0217"> Homozygous for LAL deletion (LAL^-/-) 4-week-old Donryu rats were assigned to receive recombinant human LAL or saline placebo produced in the transgenic chicken oviduct system. Wild-type, same-age littermate rats were used as controls. LAL LAL^-/-Rats administered by tail vein injection once weekly for 4 weeks (4 doses total) or every other week for 4 weeks (2 doses total), as a single dose or in two equal doses given every 30 minutes did. The dose of LAL was 1 mg / kg or 5 mg / kg. The dosing regimen is shown in Table 4. Previous experiments in animal models of enzyme replacement therapy for the treatment of lysosomal storage diseases to suppress potential anaphylactic reactions (Shull et al. (1994), Proceedings of the National Academy of Science, vol.91, p.12937; Bielicki et al. (1999), The In a procedure based on the Journal of Biological Chemistry, 274, p.36335; Vogler et al. (1999), Pediatric Research, 45, p.838) (this disclosure is incorporated herein by reference in its entirety), rat. Was pretreated with diphenhydramine (5 mg / kg).</p><p num="0218"> FIG. 29 shows the daily weight gain course of rats receiving 1 mg / kg LAL per week, 5 mg / kg LAL per week or 5 mg / kg LAL per 2 weeks. The figure shows that there is little or no difference in therapeutic effect between the two dose sizes and frequencies.</p><p num="0219"><tables num="4"><img id="000009" he="109" wi="141" file="JP2016190867A_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0220">LAL treated with recombinant LAL^-/-Pathological examination of rats At the end of the experiments described in Example 1, laboratory animals were humanely euthanized and necropsied for gross pathology, histopathology and clinical chemistry. Gross autopsy included examination of the outer surface of the body, all openings, as well as the cranial cavity, thoracic cavity, abdominal cavity and their contents. The mass of the internal organs and tissues of the rat was determined, and the organs and tissues were collected and fixed with 10% neutral buffered formalin. After fixation, tissues were treated to prepare slides of hematoxylin / eosin stained sections and evaluated.</p><p num="0221"> Macroscopic pathological examination of the treated individuals analyzed showed that liver size and color were fairly normal, as seen in the autopsy of FIG. Determined the organ-to-body weight ratio, which resulted in a reduction in the relative organ size of the liver, spleen, mesenteric tissue, duodenum, jejunum and ileum of successfully treated dissected individuals compared to placebo-treated rats. Shown (Fig. 23). The histopathology of the LAL liver tissue of the treated rats analyzed showed a essentially normal liver histology, showing a sharp contrast to placebo-treated rats with significant accumulation of foamy macrophages. (Fig. 30).</p><p num="0222">Example 18 Treatment of Wolman's disease (WD) with recombinant LAL Because it is difficult to gain weight after birth and growth is poor, a girl patient is hospitalized 7 weeks after birth. On initial physical examination, the patient weighs 3.6 kg (3.7 kg at birth) and is thin, with wrinkles due to cutis laxa. The abdomen is swollen, with a 6 cm hard hepatomegaly and about 4 cm hard splenomegaly. Lymph node hypertrophy is observed in the inguinal region, and muscle activity is weak.</p><p num="0223"> The initial hemoglobin level is 9.2 gm, the platelet count is 506,000 and the white blood cell is 11,550. Urinalysis is normal and bone marrow smears show vacuoled lymphocytes and numerous foam cells. Hematological measurements are total lipid 834 mg / 100 ml, phospholipid 176 mg / 100 ml, triglyceride 141 mg / 100 ml, cholesterol 129 mg / 100 ml, bilirubin 0.3 mg / 100 ml, alkaline phosphatase 9.0 BU%, SGOT 90 units. , SGPT is 50 units, choline esterase is 20 units, urea nitrogen is 8.3 mg, and fasting blood glucose is 45 mg / 100 ml. Abdominal CT scan shows bilateral adrenal hypertrophy with hepatosplenomegaly and calcification.</p><p num="0224"> The patient is surgically implanted with a venous access port for medication. After connecting the port to a portable injector, patients are pretreated with 1 mg / kg diphenhydramine 20 minutes prior to LAL infusion to reduce possible anaphylactic infusion reactions. LAL is then administered to the patient at 1 mg / kg by intravenous injection over 5 hours. This treatment is repeated once every 7 days indefinitely.</p><p num="0225"> Within 2 weeks of the first LAL administration, the patient experiences significant improvement in weight gain and normalization of the size of important abdominal organs as determined by ultrasound. Test results show that injection of LAL restored the patient's lysosomal acid lipase activity and corrected the associated abnormalities.</p><p num="0226">Example 19 Treatment of Cholesteryl Ester Accumulation (CESD) with Recombinant LAL A 3-year-old boy with an pruritic abdominal rash is examined by his pediatrician. Examination of the abdomen reveals hepatomegaly by a doctor and is confirmed by ultrasound. No diagnosis is made at this point and the patient is monitored regularly.</p><p num="0227"> At the age of eight, the patient is hospitalized for gastroenteritis. Optical microscopy of liver biopsy shows an increase in intracytoplasmic glycogen and small lipid droplets in hepatocytes. Electron microscopy reveals membrane-enclosed lipid droplets with small, high-electron density granules. A working diagnosis of type III glycogen accumulation disease (debranching enzyme deficiency) is made, but the debranching enzyme activity of skin fibroblasts is normal.</p><p num="0228"> Hepatomegaly continued at age 10 and a second liver biopsy was performed. Light microscopy shows changes in the lobular structure of the liver parenchyma, hepatocytes swelling with cytoplasmic granules and vacuoles, accompanied by mild periportal fibrosis. The acid lipase activity of fibroblasts was found to be 7% of normal, confirming the diagnosis of CESD. Total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) have plasma concentrations of 7.51, 3.24, and 5.58 mmol / L, respectively, above the 95th percentile of their age and gender, and plasma. Medium-density lipoprotein cholesterol (HDL-C) is below the 5th percentile at 0.47 mmol / L, and patients have complex hyperlipidemia (hypercholesterolemia, hypertriglyceridemia, low alpha lipoproteinemia and Hyperbeta lipoproteinemia).</p><p num="0229"> The patient is surgically implanted with a venous access port for medication. After connecting the port to a portable injector, patients are pretreated with 5 mg / kg diphenhydramine 20 minutes prior to LAL infusion to reduce possible anaphylactic infusion reactions. LAL is then administered to the patient at 5 mg / kg by intravenous injection over 5 hours. This treatment is repeated once every 14 days indefinitely.</p><p num="0230"> Within 2 weeks of the first LAL administration, the patient experiences significant improvement in weight gain and normalization of the size of important abdominal organs as determined by ultrasound. Test results show that injection of LAL restores the patient's lysosomal acid lipase activity and corrects the associated abnormalities.</p><p num="0231">Example 20 Description and composition of the drug The drug substance of LAL (SBC-102) described herein is recombinant human lysosomal acid lipase (rhLAL) purified from egg white produced by transgenic chickens. The additives used in SBC-102 are similar to those used in other products for lysosomal storage disorders (LSD) currently on the market and have been selected to maintain the stability of the formulation. There is.</p><p num="0232"> SBC-102 is a clear, colorless, sterile liquid provided in a clear I-type borosilicate glass vial with FluroTec® coated non-natural latex (butyl) stopper and aluminum crimp seal. Will be done. SBC-102 is SBC-102 (2 mg / mL), trisodium citrate dihydrate (13.7 mg / mL, USP), citrate monohydrate (1.57 mg / mL, USP), human serum albumin ( It is provided as an aqueous solution consisting of 10 mg / mL (USP) and water for injection (final body integral, USP). The pH of SBC-102 is 5.9 ± 0.2. SBC-102 does not contain preservatives and the vial is intended for single use.</p><p num="0233"><tables num="5"><img id="000010" he="27" wi="141" file="JP2016190867A_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0234">Ingredients of the formulation<tables num="6"><img id="000011" he="36" wi="99" file="JP2016190867A_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0235"> Each example of the above specification is provided for explaining the present invention, and does not limit the present invention. In fact, it will be apparent to those skilled in the art that various modifications, combinations, additions, deletions and changes in the present invention may be made without departing from the scope or gist of the present invention. For example, features described or described as part of one embodiment can be used in another embodiment to provide additional embodiments. The present invention is intended to include such modifications, combinations, additions, deletions and changes.</p><p num="0236"> All documents cited in the above specification (eg, US patents, US patent applications, publications) are incorporated herein by reference. Various modifications and modifications of the present invention will be apparent to those skilled in the art without departing from the scope and gist of the present invention.</p><p num="0237"> Although the present invention has been specifically shown and described with reference to an exemplary embodiment, those skilled in the art will not deviate from the scope of the invention as included in the appended claims. It will be appreciated that various modifications of form and detail can be made in the invention.</p>