RU2575074C2 - Lysosomal storage disorder enzyme - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biochemistry and represents a pharmaceutical compound for treating conditions caused by lysosomal acid lipase deficit, containing recovered human recombinant lysosomal acid lipase including SEQ ID NO: 2, and a pharmaceutically acceptable carrier, solvent or excipient, wherein the pharmaceutical compound represents an aqueous solution, which has pH from approximately 5.6 to approximately 6.2, or pH 5.9±0.2, and LAL is N-attached glycolised in position Asn¹⁵, Asn⁸⁰, Asn¹⁴⁰, Asn²⁵² and Asn³⁰⁰ of the sequence SEQ ID NO: 2 and contains certain N-linked glycosylation profile.

EFFECT: invention enables producing the effective preparation for treating conditions caused by lysosomal acid lipase deficit.

20 cl, 30 dwg, 6 tbl, 20 ex

Description

RELATED APPLICATIONS

This application claims priority over provisional patent application US No. 61/343177 dated April 23, 2010, provisional patent application US No. 61/396376 of May 26, 2010, provisional patent application US No. 61/403011 of September 9, 2010, provisional patent application US No. 61/456014 of October 29, 2010, provisional application for US patent No. 61/432372 dated January 13, 2011. The entire contents of the above documents are hereby incorporated by reference in their entirety.

KNOWN LEVEL OF TECHNOLOGY

Lysosomal acid lipase deficiency (LAL) is a very rare lysosomal storage disease (LBI), characterized by the absence of decay of cholesterol esters (EC) and triglycerides (TG) in lysosomes due to lack of enzyme. LAL deficiency resembles other lysosomal storage diseases with accumulation of substrate in a number of tissues and cells of various types. With a lack of LAL, substrate accumulation is most pronounced in the cells of the reticuloendothelial system, including Kupffer cells in the liver, histiocytes in the spleen, and cells of the lamina propria lamina. Reticuloendothelial cells express a macrophage mannose / N-acetylglucosamine receptor (also known as macrophage mannose receptor or MMR, CD206), which mediates the binding, cell uptake and lysosomal internalization of proteins with N-glycans that end up with mannose or GlcNAc, providing a pathway for potential correction of enzyme deficiency in such key cell types.

LAL deficiency is a multisystem disease, which most often manifests itself in the form of complications from the gastrointestinal tract, liver and cardiovascular system and is associated with significant mortality and morbidity. The clinical effects of LAL deficiency are caused by a massive accumulation of lipid material in lysosomes in a number of tissues, as well as a significant violation of the mechanisms of lipid and cholesterol homeostasis, including a significant increase in liver cholesterol synthesis. LKL deficiency has at least two phenotypes, represented by Wolman’s disease (BV) and cholesterol ester accumulation disease (BNEC).

Wolman's disease is the most aggressive manifestation of LAL deficiency. This phenotype is characterized by manifestations from the liver and gastrointestinal tract, including impaired growth, impaired absorption, steatorrhea, significant weight loss and hepatomegaly. Wolman's disease is a rapidly progressive and fatal disease that usually occurs during the first year of life. A review of reported cases indicates that survival rates after 12 months of age are extremely unusual for patients with growth disorders caused by LAL deficiency during the first year of life. In this more aggressive form, dysplasia is a dominant clinical sign and is a key factor in early mortality. Liver involvement is determined by an increase in the liver and an increase in transaminases, which is very common in infants. Physical indicators include bloating accompanied by hepatomegaly and splenomegaly, and an X-ray often reveals adrenal calcification. Laboratory tests usually reveal elevated levels of transaminases in serum and the absence or significant decrease in the activity of the enzyme LAL. Also, patients have elevated levels of cholesterol and triglycerides in the blood.

Currently used methods of treating Wolman's disease are extremely limited. Antibiotics are administered to infants with pyrexia and / or a proven presence of infection. Steroid replacement therapy may be prescribed to treat adrenal insufficiency and specialized parenteral nutrition, and in the absence of evidence that such interventions prevent death, it is also unclear about their effect on short-term survival. Four patients with LAL deficiency were treated and bone marrow transplantation: all four patients died due to complications from the procedure within a few months after transplantation.

In patients with LAL deficiency, the predominant lesions of the liver and cardiovascular system may later manifest themselves, which is often called the disease of accumulation of cholesterol esters (BNEC). Serious disorders with significant hepatomegaly, hepatocyte necrosis, an increase in transaminases, cirrhosis and fibrosis are noted in the liver with BNEC. Due to elevated EC and TG levels, with LAL deficiency, hyperlipidemia and accelerated atherosclerosis are also noted. In particular, at an early age, the accumulation of fatty deposits in the walls of arteries is described. Such deposits narrow the arterial lumen, which can lead to vascular occlusion and an increased risk of significant cardiovascular events, including myocardial infarction and stroke. The manifestation of BNEC varies greatly in some patients who were not diagnosed before the onset of complications in adulthood, while in others, liver dysfunction manifests itself already in early childhood. BNECh causes short life expectancy and essentially poor health; life expectancy in patients with BNEC depends on the severity of the associated complications.

Currently used methods for treating the BNEC phenotype are focused on controlling lipid accumulation through a diet that excludes foods high in cholesterol and triglycerides, suppressing cholesterol synthesis and producing apolipoprotein B through the administration of drugs that lower cholesterol levels. Despite the fact that certain clinical improvements may be noted, the background manifestations of the disease persist, and disease progression is noted.

In most cases, treatment of LAL deficiency requires long-term treatment. In addition, due to the high cost of protein therapeutics, it is advisable to administer a minimally effective amount of the drug to treat LK deficiency. However, to date, there is no effective therapy for the treatment of LAL deficiency, in particular for patients suffering from Wolman disease and BNEC. Therefore, there is an urgent need for effective therapy with a minimum frequency of administration to improve the quality of life of patients. There is also a need to create a platform for high expression and robust protein production, which can ensure the production of LCL proteins that are stable and efficiently directed relative to the lysosomal compartment in the cells of affected tissues in patients.

SUMMARY OF THE INVENTION

This application describes compositions with LAL, which are particularly suitable for use in the treatment of, for example, conditions caused by LAL deficiency. The LCL molecules described herein contain individual polysaccharide structures, which ensures efficient and rapid uptake of cells in lysosomes when administered to a subject, such as a human subject.

In one aspect, the compositions described herein include human LAL, with a significant percentage of human LAL containing at least one mannose-6-phosphate glycan molecule that serves as a ligand for internalization of the mannose-6-phosphate receptor on the surface of cells located for example, on hepatocytes. In one embodiment, 30% or more, for example at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least at least 90%, at least 95%, at least 97%, or at least 99% of the LCL contained in the composition contains at least one mannose-6-phosphate molecule. The mannose-6-phosphate molecule may be, for example, on an N-glycan structure located on one or more residues selected from the group consisting of Asn ¹⁵ , Asn ⁵¹ , Asn ⁸⁰ , Asn ¹⁴⁰ , Asn ²⁵² and Asn ³⁰⁰ with the sequence SEQ ID NO: 2.

In another aspect of the invention, the compositions described herein include human LAL, with a significant percentage of human LAL not containing a sialic acid molecule in any of its N-glycan structures, which can sometimes affect the internalization of enzymes in cells. In one embodiment, 15% or less, for example 10% or less, 5% or less, 2% or less, 1% or less, or essentially no LCL contained in the composition contains a sialic acid molecule in any of their N-glycan structures.

In another aspect of the invention, the compositions described herein include human LAL, with a significant percentage of human LAL not containing a fucose molecule in any of its N-glycan structures. In one embodiment, the invention is 50% or less, for example 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 2% or less, 1% or less, or essentially none single LCL contained in the composition does not contain a fucose molecule in any of its N-glycan structures.

In yet another aspect of the invention, vectors, host cells, expression systems, and corresponding methods suitable for producing compositions containing LAL are described.

Typically, the LCL of the invention described and discussed in the text of this application is represented by human LCL. In one embodiment, a composition comprising LKL comprises mature LKL with the following amino acid sequence:

SGGKLTAVDPETNMNVSEIISYWGFPSEEYLVETEDGYILCLNRIPHGRKNHSDKGPKPVVFLQHGLLADSSNWVTNLANSSLGFILADAGFDVWMGNSRGNTWSRKHKTLSVSQDEFWAFSYDEMAKYDLPASINFILNKTGQEQVYYVGHSQGTTIGFIAFSQIPELAKRIKMFFALGPVASVAFCTSPMAKLGRLPDHLIKDLFGDKEFLPQSAFLKWLGTHVCTHVILKELCGNLCFLLCGFNERNLNMSRVDVYTTHSPAGTSVQNMLHWSQAVKFQKFQAFDWGSSAKNYFHYNQSYPPTYNVKDMLVPTAVWSGGHDWLADVYDVNILLTQITNLVFHESIPEWEHLDFIWGLDAPWRLYNKIINLMRKYQ (SEQ ID NO: 2).

In another embodiment, mature LCL has the following amino acid sequence:

GKLTAVDPETNMNVSEIISYWGFPSEEYLVETEDGYILCLNRIPHGRKNHSDKGPKPVVFLQHGLLADSSNWVTNLANSSLGFILADAGFDVWMGNSRGNTWSRKHKTLSVSQDEFWAFSYDEMAKYDLPASINFILNKTGQEQVYYVGHSQGTTIGFIAFSQIPELAKRIKMFFALGPVASVAFCTSPMAKLGRLPDHLIKDLFGDKEFLPQSAFLKWLGTHVCTHVILKELCGNLCFLLCGFNERNLNMSRVDVYTTHSPAGTSVQNMLHWSQAVKFQKFQAFDWGSSAKNYFHYNQSYPPTYNVKDMLVPTAVWSGGHDWLADVYDVNILLTQITNLVFHESIPEWEHLDFIWGLDAPWRLYNKIINLMRKYQ (SEQ ID NO: 3).

In another embodiment, mature LCL has the following amino acid sequence:

TAVDPETNMNVSEIISYWGFPSEEYLVETEDGYILCLNRIPHGRKNHSDKGPKPVVFLQHGLLADSSNWVTNLANSSLGFILADAGFDVWMGNSRGNTWSRKHKTLSVSQDEFWAFSYDEMAKYDLPASINFILNKTGQEQVYYVGHSQGTTIGFIAFSQIPELAKRIKMFFALGPVASVAFCTSPMAKLGRLPDHLIKDLFGDKEFLPQSAFLKWLGTHVCTHVILKELCGNLCFLLCGFNERNLNMSRVDVYTTHSPAGTSVQNMLHWSQAVKFQKFQAFDWGSSAKNYFHYNQSYPPTYNVKDMLVPTAVWSGGHDWLADVYDVNILLTQITNLVFHESIPEWEHLDFIWGLDAPWRLYNKIINLMRKYQ (SEQ ID NO: 4).

In another embodiment, mature LCL has the following amino acid sequence:

AVDPETNMNVSEIISYWGFPSEEYLVETEDGYILCLNRIPHGRKNHSDKGPKPVVFLQHGLLADSSNWVTNLANSSLGFILADAGFDVWMGNSRGNTWSRKHKTLSVSQDEFWAFSYDEMAKYDLPASINFILNKTGQEQVYYVGHSQGTTIGFIAFSQIPELAKRIKMFFALGPVASVAFCTSPMAKLGRLPDHLIKDLFGDKEFLPQSAFLKWLGTHVCTHVILKELCGNLCFLLCGFNERNLNMSRVDVYTTHSPAGTSVQNMLHWSQAVKFQKFQAFDWGSSAKNYFHYNQSYPPTYNVKDMLVPTAVWSGGHDWLADVYDVNILLTQITNLVFHESIPEWEHLDFIWGLDAPWRLYNKI INLMRKYQ (SEQ ID NO: 19).

In another embodiment, the mature LCL 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.

The invention also describes compositions that contain isolated mixtures of useful individual protein molecules of the individual type, such as the proteins described herein, wherein one or more protein molecules contained in the mixture has a specific oligosaccharide structure attached, in particular, to the oligosaccharide structure described herein. For example, the invention describes isolated mixtures of LCL molecules, for example human LCL molecules, which contain a LCL molecule glycosylated by one or more of the following structures from A-n to O-n:

Square = N-Acetylglucosamine

Black square = mannose-6-phosphate

Circle = Mannose

Black circle = galactose

Black Triangle = Fucose

According to one aspect of the present invention, the composition includes any isolated single or combination of the polypeptides described above. In one embodiment of the invention, the composition may be a pharmaceutical composition, for example, a composition that further includes pharmaceutically acceptable carriers, such as a composition, for example, suitable for administration to a subject (for example, a person, in particular a patient suffering from or having a diagnosis of a certain condition) . The composition may be administered using a variety of routes, including intravenous administration. In another embodiment, the composition may further include a second agent. Such an agent can be represented by a medicament or an agent that can influence or modify a biological process when administered to a subject. For example, the second agent may be an immunomodulator. Such immunomodulatory agents may include any agent that, when administered together (i.e., administered at the same time or shortly before or after) with any of the LCL compositions described herein, can affect or reduce the immunogenicity of the LCL composition in a subject (e.g., rituximab or any B cell-reducing antibody).

In a final aspect of the invention, methods and compositions for the treatment of symptoms associated with LAL deficiency are described.

Additional objects and aspects of the present invention will become more apparent after reviewing the detailed description presented below in conjunction with the accompanying figures and sequences.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

FIG. 1 depicts the amino acid sequences of human LAL. The amino acid sequence of the recombinant hLKL is 100% homologous to the human LKL sequence found in nature. The mature form of hLKL is emphasized.

FIG. 2 depicts the nucleotide sequence of recombinant hLKL, rhLKL transgene pALVIN-OVRl-I-hLAL-dSA.

FIG. 3A and 3B show pALVIN-OVRl-I-hLAL-dSA and proviral plots. FIG. 3A is a diagram of a human LCR expression retroviral vector used in the production of transfected particles (plasmid DNA sequence is given in Appendix A). FIG. 3 depicts the proviral site of pALVIN-OVRl-I-hLAL-dSA that has been integrated into the genome. SIN LTR - self-inactivating long terminal repeat; OV DHSIII - enhancer; DNase - hypersensitive plot III of the egg albumin gene; OV is the intron; 5 'untranslated section of egg albumin and intron 1; hLAL — human LKL cDNA; OV 3 'UTR - 3' untranslated portion of the egg albumin gene; partial gag - partial gag gene; LTR - long terminal repeat.

FIG. 4 depicts the nucleotide sequence of pALVIN-OVRl-I-hLAL-dSA.

FIG. 5 depicts the nucleotide sequence of the proviral region of pALVIN-OVRl-I-hLAL-dSA that has been integrated into the genome.

FIG. 6 depicts the nucleotide sequence of the vector pALVIN-OV-l.l-I.

FIG. 7 depicts the nucleotide sequence of the rhLAL adapter.

FIG. 8 depicts the nucleotide sequence of rhLAL, including a partial egg albumin promoter.

FIG. 9 depicts the nucleotide sequence of the OVR1 promoter.

FIG. 10 schematically depicts the steps used to construct the vector pALVIN-OVR1 -I-hLAL-dSA.

FIG. 11 depicts real-time PCR analysis of blood DNA samples obtained from hemizygous transgenic progeny of G1 XLL109. Signals from duplicate hemizygous G1 1LL7466 progenic DNA samples are indicated by curves that initiate an increase in Rn delta before cycle 22. Curves for two non-transgenic genes are shown; such curves remain at or near the baseline for at least 34 cycles.

FIG. 12A-D depict Southern analysis of G1 chickens carrying the ALVIN-OVRl-I-hLAL-dSA transgene. FIG. 12A illustrates an integrated transgene pattern and flanking genomic regions with a known BlpI transgene site position and a predicted position of BlpI flanking genomic sites. The positions of the OV promoter probe and the hLAL coding sequence probe (hLAL probe) are indicated by black bars. The positions of the 4.3 kb and 10.6 kb bands determined during the Southern analysis are presented along with the predicted sizes of the genomic and transgenic sections of the 4.3 kb and 10.6 kb bands. FIG. 12B depicts Southern blotting of genomic DNA digested with BlpI and probed with an OV probe. WT CTRL is genomic DNA isolated from non-transgenic chicken. G1 transgene ID numbers are shown above the bands. The position and size (kb) of the molecular weight of the markers is presented on the left side of the blot. The position and size of a specific transgene fragment (4.3 kb) and the endogenous egg albumin gene (4.1 kb) are presented on the right side of the blot. FIG. 12C depicts Southern blotting with an hLAL probe. The position and size of a specific transgene fragment (10.6 kb) is presented to the right of blotting. FIG. 12D shows a portion of the image of FIG. 12B on a larger scale to represent the presence of bands of 4.1 and 4.3 kb.

FIG. 13A depicts the ALVIN-OVRl-I-hLAL-dSA transgene scheme. Also shown is the size of the ApaLI bands, which are predicted to be determined by the OV probe and the hLAL probe. FIG. 13B shows an ALVIN-OVRl-I-hLAL-dSA transgene southern blot analysis assay for confirming the size of the transgene.
Southern blotting of genomic DNA cleaved with ApaLI using an OV probe (left panel) or an hLAL probe (right panel). WT CTRL is genomic DNA isolated from non-transgenic chicken. Above each bar is an ID number for Gl. The position and size (kb) of the molecular weight of the markers is presented on the left side of the blot. The position and size of a specific transgene fragment (OV promoter probe, 3.6 kb; hLAL probe, 3.8 kb) and endogenous egg albumin gene (7.7 kb) are presented on the right side of the blot.

FIG. 14 shows the cell line of transgenic chickens. For each chicken, the generation number (G0, G1 or G2), identification number, gender and hatch date are indicated. All chickens, except generation G1, belong to other lines.

FIG. 15 depicts the steps for purifying hLAL from egg white.

FIG. 16 depicts N-glycans contained in an N-linked glycosylated structure in LCL prepared in accordance with this invention. Square - N-acetylglucosamine; black square - mannose-6-phosphate; circle - mannose; black circle - galactose; the black triangle is fucose.

FIG. 17 shows the relative position of the predicted N-glycan sites indicated for the LCL polypeptide (arrow) with the sequence SEQ ID NO: 1. Presents N-glycans, which are structural representatives defined for each site. Square - N-acetylglucosamine; black square - mannose-6-phosphate; circle - mannose; black circle - galactose; the black triangle is fucose.

FIG. 18 depicts phosphorylated N-glycans obtained by PNGase and analyzed by MALDI-TOF. Structures are indicated.

FIG. 19 depicts the effect of dephosphorylation of LAL on the retention time of HPAEC-PAD relative to N-glycans. The LAL obtained according to the invention was dephosphorylated by bacterial alkaline phosphatase (upper panel) or not exposed (lower panel). The resulting N-glycans were analyzed using HPAEC-PAD.

FIG. 20 depicts the colocalization of recombinant human LAL (SBC-102) and a lysosomal marker in lysosomes of cells examined by confocal fluorescence microscopy and sequential scan mode.

FIG. 21 depicts the binding specificity of recombinant human LAL (SBC-102) with the GlcNAc / mannose receptor, which was evaluated in competitive binding assays using the macrophage cell line NR8383.

FIG. 22 depicts the activity of recombinant human LAL in cells with normal LAL content and in vitro LAL deficiency.

FIG. 23 depicts the effect of treatment with recombinant human LAL (SBC-102) on the mass of internal organs in rats with LAL deficiency. The size of the organs is presented as a percentage of body weight, determined at the age of 8 weeks in rats LKL ^{- / -} and LKL ^{+ / +} after weekly administration of a carrier or SBC-102 at a dose of 5 mg / kg for 4 weeks.

FIG. 24 shows the body weight in wild type rats and rats with deficiency LKL weekly after administration of vehicle or SBC-102 at a dose of 5 mg / kg ^-1 for 4 weeks. The dose is indicated on the X axis in the form of rhombuses, starting from 4 weeks.

FIG. 25 shows liver cholesterol, cholesterol esters, and triglycerides at 8 weeks of age in WT rats and LKL-deficient rats after weekly administration of vehicle or recombinant human LKL (SBC-102) at a dose of 5 mg / kg ^-1 for 4 weeks.

FIG. Figure 26 shows the percent increase in body weight in rats with LAL deficiency after 4 weeks of administration of recombinant human LAL (SBC-102) at the indicated levels and schedule, as determined at 8 weeks of age.

FIG. 27 shows the liver weight as a percentage of body weight in rats with LAL deficiency after 4 weeks of administration of SBC-102 at the indicated levels and schedule, as determined at 8 weeks of age.

FIG. 28 shows cholesterol ester levels in tissues of rats with LAL deficiency after 4 weeks of administration of SBC-102 at the indicated levels and schedule, as determined at 8 weeks of age.

FIG. Figure 29 shows the daily progress in weight gain in rats given 1 mg / kg LKL per week or 5 mg / kg LKL per week or 5 mg / kg LKL once every two weeks.

FIG. 30 depicts a complete anatomical and pathological examination of the treated animals, showing essentially normalization of liver size and color during dissection (upper part), as well as histopathological analysis of liver tissue for LCL in the treated rats, which showed normal histological parameters of the liver in stark contrast relatively significant accumulation of foamy macrophages in animals treated with placebo (lower part).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The text of this application provides certain terms to illustrate and determine the meaning and scope of various concepts used to describe the present invention.

In the context of this invention, the term “acceptable” with respect to a composition, composition or ingredient means the absence of any permanent deleterious effect on the general health of the subject being treated.

In the context of this invention, the term “administration” or “administer” means the provision of the recombinant lysosomal acid lipase of the invention to a subject in need of treatment.

The term "nucleic acid or polynucleotide sequence" includes, but is not limited to, sequences of eukaryotic mRNA, cDNA, genomic DNA, synthesized DNA and RNA, including naturally occurring nucleotide bases such as adenine, guanine, cytosine, thymidine and uracil. The term also includes sequences with one or more modified bases.

The term "bird" in the context of this invention refers to any species, subspecies or race of the organism from the taxonomic class of birds, such as, but not limited to, chicken, turkey, duck, goose, quail, pheasants, parrots, finches, hawks, crows and ratites such as ostriches, emus and cassowary. The terms include various known breeds of Gallus gallus, or chickens (for example, white leggorn, brown leggorn, striped plymouth rock, Sussex, New Hampshire, Rhode Island, Australorp, Minor, Amrox, California Gray), as well as turkey, pheasant, quail breeds , ducks, ostriches and other poultry that are bred on a commercial basis. They also include a separate bird organism at all stages of development, including the stages of the embryo and the fetus.

The term “therapeutic proteins” or “pharmaceutical proteins” includes an amino acid sequence that, in whole or in part, constitutes a preparation.

The term “coding sequence” or “open reading frame” means a polynucleotide or nucleic acid sequence that can be transcribed and translated (in the case of DNA) or translated (in the case of mRNA) into an in vitro or in vivo polypeptide by monitoring the corresponding regulatory sequences. The boundaries of the coding sequence are determined by the start codon of translation at the 5 '- (amino) end and the stop codon of translation at the 3' - (carboxyl) end. The transcription termination sequence is usually located at the 3'-end of the coding sequence. The coding sequence can be flanked at the 5'- and / or 3'-ends with untranslated regions.

The term "exon" refers to the part of a gene that, when transcribed in a nuclear transcript, is "expressed" in the cytoplasmic mRNA after removal of introns or insertion sequences by nuclear splicing.

The terms “control sequences” or “regulatory sequences” of nucleic acids mean promoter sequences, start and stop translation codons, ribosome binding sites, polyadenylation signals, transcription termination sequences, “left” regulatory domains, enhancers, etc., as necessary and sufficient for transcription and translation of a separate coding sequence in a particular host cell. Examples of control sequences suitable for eukaryotic cells are represented by promoters, polyadenylation signals, and enhancers. It is not necessary that all such control sequences are present in a recombinant vector, since they are necessary for transcription and translation of a particular gene present.

The term "functionally or operatively linked" refers to the configuration of the coding and control sequences to perform the desired function. Thus, control sequences operably linked to a coding sequence are capable of influencing the expression of a coding sequence. The coding sequence is operably linked or under the control of the transcriptional regulation region into the cell when the DNA polymerase binds to the promoter sequence and transcribes the coding sequence into mRNA, which can be transcribed into the encoded protein. Control sequences need not be adjacent to the coding sequence because they act as a regulator of expression. Thus, for example, between a promoter sequence and a coding sequence there may be untranslated but transcribed sequences, and the promoter sequence may be considered “functionally linked” to the coding sequence.

The terms “heterologous” and “exogenous” with respect to nucleic acid sequences, such as coding sequences and control sequences, denote sequences that are not normally associated with a region of a recombinant vector or with a single chromosome locus, and / or are not normally associated with a single cell. Thus, the “exogenous” portion of a nucleic acid vector is represented by an identifiable nucleic acid segment within or attached to another nucleic acid molecule that is not in nature in association with another molecule. For example, an exogenous region of a vector may include a coding sequence flanked by sequences that are not associated with the coding sequence in nature. Another example of an exogenous coding sequence is represented by a vector in which the coding sequence is not present in nature (for example, is represented by a synthetic sequence with codons different from the native gene). Similarly, a host cell transformed with a vector or nucleic acid not normally found in the host cell will be considered exogenous for the purposes of this invention.

In the context of this invention, the terms "N-glycan", "oligosaccharide", "oligosaccharide structure", "glycosylation pattern", "glycosylation profile" and "glycosylation structure" have essentially the same meaning and each term denotes one or more structures which are formed from sugar residues and are attached to glycosylated proteins.

The term "exogenous protein" in the context of this invention refers to a protein that is not naturally present in a single tissue or cell, and such a protein is an expression product of an exogenous expression vector or transgene, or is a protein that is not naturally present in a given amount in a single tissue or the cage. A protein that is exogenous in an egg is represented by a protein that is not normally present in the egg. For example, an exogenous protein for an egg may be a protein that is present in an egg as a result of expression of a coding sequence present in a transgene of an egg-laying animal.

"Endogenous gene" means a naturally occurring gene or fragment thereof, which is usually associated with a single cell.

The term “LKL” means “human lysosomal acid lipase”, “SBC-102”, or “human lysosomal acid lipase molecule”, and such terms are used interchangeably herein.

The expression products described herein may consist of a protein material with a specific chemical structure. However, the exact structure depends on a number of factors, in particular chemical modifications characteristic of proteins. For example, since all proteins contain ionizable amino and carboxyl groups, the protein can be obtained in the form of an acidic or basic salt, or in a neutral form. The primary amino acid sequence can be obtained using sugar molecules (glycosylation) or other chemical methods, including covalent or ionic addition, for example, lipids, phosphate, acetyl groups and the like, which is often found when combined with saccharides. Such modifications may occur in vitro or in vivo, the latter occurring in the host cell via post-translational processing systems. Such modifications may increase or decrease the biological activity of the molecule, and such chemically modified molecules are also included in the scope of this invention.

Alternative methods for cloning, amplification, expression and purification will become apparent to those skilled in the art. Representative methods are described in Sambrook, Fritsch, and Maniatis, Molecular Cloning, a Laboratory Manual, 2 ^nd Ed., Cold Spring Harbor Laboratory (1989).

The term "vector" refers to a polynucleotide comprising a single-stranded, double-stranded, circular or supercoiled DNA or RNA. A typical vector may consist of the following elements that are functionally linked at appropriate distances to ensure expression of a functional gene: replication origin, promoter, enhancer, 5 'mRNA leader sequence, ribosome binding site, nucleic acid cassette, termination and polyadenylation sites and selectable marker sequences . One or more of these elements may be described in separate patent applications. The nucleic acid cassette may include a restriction site for insertion of an expressed nucleic acid sequence. In a functional vector, the nucleic acid cassette contains the nucleic acid sequence to be expressed, including translation start and termination sites. In some cases, an intron may be included in a vector, for example, from the end of 5 'relative to the coding sequence. The vector is designed in such a way that a separate coding sequence is located in the vector with the corresponding regulatory sequences, and the location and orientation of the coding sequence relative to the control sequences is such that the control sequence is transcribed under the “control” of the control or regulatory sequences. Modification of sequences encoding a single protein may be desirable to achieve this end. For example, in some cases it may be necessary to modify the sequence so that it is attached to the control sequence with the appropriate orientation; or to maintain a reading frame. Control sequences and other regulatory sequences may be ligated to the coding sequence before insertion into the vector. Alternatively, a coding sequence can be cloned directly into an expression vector that already contains control sequences and a corresponding restriction site that is within the reading frame and under control of the control sequences.

The term "promoter" refers to the region on the DNA to which RNA polymerase binds to initiate transcription of the gene. In some embodiments, the promoter may be modified by adding or deleting sequences, or replacing with alternative sequences, including naturally occurring or synthesized sequences, as well as sequences that can be represented by a combination of synthesized and naturally occurring sequences. Most eukaryotic promoters include two types of recognition sequences: a TATA box and “left” promoter elements. The former are located upstream of the transcription initiation site and are involved in the direction of RNA polymerase to initiate transcription in the right place, while the latter determine the transcription rate and are higher relative to the TATA box. Enhancer elements can also stimulate transcription from attached promoters, but most function exclusively in a single cell type. Many elements of the enhancer / promoter derived from viruses, for example, the SV40 promoter, the cytomegalovirus (CMV) promoter, the Routh sarcoma virus (RSV) promoter, and the murine leukemia virus (MLV) promoter, are active in a variety of cell types and are called ubiquitous. On the other hand, unconstitutional promoters, such as the promoter of the mouse breast tumor virus (MMTV), can also be used in this invention. The nucleic acid sequence inserted into the cloning site can have any open reading frame encoding the polypeptide of interest, provided that the coding sequence encodes the polypeptide of interest and does not contain splice sites that can block the production of the corresponding mRNA molecules and / or produce in an aberrant manner connected or abnormal mRNA molecules.

In the context of this invention, the term "pharmaceutical composition" means a mixture of the compounds described herein with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, disintegrants and / or excipients.

The term “obtained from poultry” or “obtained from poultry” means a composition or substance obtained or produced by a bird. The term "poultry" refers to birds bred at home, including, but not limited to, chickens, ducks, turkeys, quails and ratites. For example, the term “derived from poultry” may mean descent from chickens, turkeys and / or quails.

The term “retroviral particle”, “transmitting particle” or “transduction particle” means a virus that is replication-defective or replication-competent that is capable of transferring non-viral DNA or RNA to the cell. In a separate embodiment, retroviral particles used to produce transgenic birds in accordance with the invention are prepared as described in US Pat. 7524626 dated April 28, 2009, the description of which is incorporated herein in its entirety by reference.

The terms "transformation", "transduction" and "transfection" mean the introduction of a polynucleotide into a blastoderm cell of a bird. The term "magnum" refers to the part of the oviduct between the funnel and the site of narrowing, containing tubular gland cells that synthesize and secrete egg white protein.

The term "transgene" means a heterologous nucleotide sequence inserted into the bird genome according to the invention. The term “transgene” may specifically refer to an exogenous coding sequence attached to an exogenous promoter or other regulatory sequence, the complete nucleotide sequence between two LTR retrovirus and / or LTR retrovirus and the nucleotide sequence between LTR, wherein the LTR belongs to the retrovirus used to introduce the transgene.

The term “optimized” is used in the context of an “optimized coding sequence,” with most of the frequently used codons for each individual amino acid among egg albumin of egg protein, lysozyme, ovomcoid, and ovotransferrin used to construct the optimized polynucleotide sequence of human interferon-a 2b ( IFN-a 2b), which is inserted into the vectors of this invention. More specifically, the DNA sequence for optimized human IFN-a 2b is based on the optimized chicken oviduct codon and was created using the BACKTRANSLATE program from the Wisconsin Package, version 9.1 (Genetics Computer Group Inc., Madison, Wis.) With a table of codons corresponding to egg proteins albumin, lysozyme, ovomukoid and ovotransferrin chicken (Gallus gallus). For example, the percentage utilization of the four codons of the amino acid alanine in four egg white proteins is 34% for GCU, 31% for GCC, 26% for GCA, and 8% for GCG. Thus, GCUs are used as the codon for most alanines in an optimized coding sequence. Vectors containing a gene for an optimized human protein are used to produce transgenic birds that express transgenic bird origin proteins in their tissues and eggs.

In the context of this invention, the term "subject" means mammals and non-mammals. Examples of mammals include, but are not limited to, humans, chimpanzees, monkeys, cattle, horses, sheep, goats, pigs; rabbits, dogs, cats, rats, mice, guinea pigs, and the like.

In the context of the present invention, the term “therapeutically effective amount” refers to any amount of a compound that, in comparison with a corresponding subject that has not received this amount, leads to an improvement in treatment, healing, prophylaxis or improvement in a disease, disorder or side effect, or reduction the rate of progression of the disease or disorder. The term also includes amounts effective to enhance normal physiological function.

The term “treat,” “treat,” or “treat,” refers to methods for reducing, improving, or reducing the symptoms of a disease or condition, preventing additional symptoms, improving or preventing the underlying causes of symptoms, inhibiting a disease or condition, stopping the development of a disease or condition, recovering from a disease or conditions, causing regression of a disease or condition, recovering from a condition caused by a disease or condition, or stopping symptoms of a disease or preventive condition skim and / or therapeutic manner.

COMPOSITIONS LKL

The invention is usually described by compositions comprising enzymes used to treat, for example, lysosomal storage diseases. In one aspect, the invention describes lysosomal storage disease enzymes, such as LAL, with a glycosylation regimen that internalizes the molecule into various types of cells. Such an invention describes recombinant human proteins, including LAL, in isolated or purified form. Isolation of lysosomal storage disease enzymes (such as LAL) may be accompanied by methods well known to those skilled in the art and used to purify proteins.

In one embodiment, the invention describes lysosomal storage disease enzymes, including, but not limited to, LAL with the N-linked glycosylation scheme described herein.

In one aspect, the compositions described herein include human LAL, with a significant percentage of human LAL containing a mannose-6-phosphate glycan molecule that serves as a ligand for internalization of the mannose-6-phosphate receptor on the surface of cells located, for example, on hepatocytes. In one embodiment, 30% or more, for example at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at at least 90%, at least 95%, at least 97%, or at least 99% of the LCL contained in the composition contains at least one molecule of mannose-6-phosphate. The mannose-6-phosphate molecule may be, for example, on an N-glycan structure located on one or more residues selected from the group consisting of Asn ¹⁵ , Asn ⁵¹ , Asn ⁸⁰ , Asn ¹⁴⁰ , Asn ²⁵² and Asn ³⁰⁰ with the sequence SEQ ID NO: 2. Glycan structures containing mannose-6-phosphate molecules include, for example, Gn and Hn shown in FIG. 16.

The recombinant human LAL of the present invention contains multiple N-linked carbohydrate chains (e.g., about 5 or 6 carbohydrate chains). N-linked glycosylated structures at each of five or six sites can be selected from An, Bn, Cn, Dn, En, Fn, Gn, Hn, In, Jn, Kn, Ln, Mn, Nn, and On, as shown in FIG. 16.

In addition, the invention describes a mixture of LCL molecules (for example, more than one LCL molecule may be present in the mixture, while the LCL molecules have the sequences SEQ ID NO: 2, 3, 4 and 19), while some or all of the LCL molecules have one or more glycosylated structures selected from Structures An, Structures Bn, Structures Cn, Structures Dn, Structures En, Structures Fn, Structures Gn, Structures Hn, Structures In, Structures Jn, Structures Kn, Structures Ln, Structures Mn, Structures Nn, and Structures On (Fig. 16). In one embodiment, a mixture of lysosomal acid lipase molecules is purified or isolated, for example, isolated from an egg or purified or isolated from an egg protein obtained from a transgenic bird.

The invention also describes a single LK molecule comprising Structure A-n. The invention also describes a single LK molecule including Structure B-n. The invention also describes a single LK molecule including a C-n structure. The invention also describes a single LK molecule comprising Structure D-n. The invention also describes a single LK molecule including Structure E-n. The invention also describes a single LCL molecule comprising an F-n structure. The invention also describes a single LCL molecule comprising Structure G-n. The invention also describes a single LK molecule including an H-n structure. The invention also describes a single LK molecule including Structure I-n. The invention also describes a single LK molecule including Structure J-n. The invention also describes a single LK molecule including a K-n structure. The invention also describes a single LK molecule including the L-n structure. The invention also describes a single lysosomal acid lipase molecule comprising Structure M-n. The invention also describes a single LK molecule including an N-n structure. The invention also describes a single LK molecule including an O-n structure.

The oligosaccharides N-attached to human LAL of the present invention do not contain terminal sialic acid and galactose residues. Those. only minor amounts of N-attached oligosaccharide structures are terminally sialylated with the presence of several galactose residues. In addition, terminal N-acetylglucosamine (GlcNAc) is present on the N-linked oligosaccharide structures of the LCL described herein. Thus, the LAL obtained according to this invention can be directed to cells, such as monocytes, macrophages and Kupffer cells.

One aspect of the invention describes LK compositions substantially free of sialic acid. In another aspect of the invention, the compositions described herein include recombinant human LAL, wherein a significant percentage of human LAL does not contain a sialic acid molecule in any of its N-glycan structures, which can sometimes affect the internalization of enzymes in cells. In one embodiment, 15% or less, for example, 10% or less, 5% or less, 2% or less, 1% or less, or essentially no LCL contained in the composition does not contain a sialic acid molecule in any from their N-glycan structures.

In another embodiment, about 95% or more of the N-attached oligosaccharides are present on a single LAL molecule of the invention without sialic acid. In another embodiment, approximately 90% or more of the N-attached oligosaccharides are present on a single LAL molecule of the invention without sialic acid. In another embodiment, about 80% or more of the N-attached oligosaccharides are present on a single LAL molecule of the invention without sialic acid. In another embodiment, more than about 70% or more of the N-attached oligosaccharides is present on a single LAL molecule of the invention without sialic acid.

In yet another embodiment of the invention, substantially none of the types of N-attached to the LAL molecules of the oligosaccharide structures of the invention contains sialic acid. In another embodiment, about 90% or more of the N-attached oligosaccharides are linked to the LAL molecule of the invention and are free of sialic acid. For example, if 20 types of oligosaccharide structures are present, 18 or more structural types do not contain sialic acid. In another embodiment, about 80% or more of the N-attached oligosaccharides are linked to the LAL molecule of the invention and are free of sialic acid. In another embodiment, about 70% or more of the N-attached oligosaccharides are linked to the LAL molecule of the invention and are free of sialic acid. In another embodiment, about 60% or more of the N-attached oligosaccharides are linked to the LAL molecule of the invention and are free of sialic acid. In another embodiment, about 50% or more of the N-attached oligosaccharides are linked to the LAL molecule of the invention and are free of sialic acid.

According to one aspect of the invention, the LCL described herein contains high levels of terminal N-acetylglucosamine. In one aspect, about 95% or more of the N-attached oligosaccharides is present on a single LAL molecule of the invention and contains terminal N-acetylglucosamine. In one embodiment, about 90% or more of the N-attached oligosaccharides are present on a single LAL molecule of the invention and contain terminal N-acetylglucosamine. In another embodiment, about 80% or more of the N-attached oligosaccharides is present on a single LAL molecule of the invention and contains terminal N-acetylglucosamine. In another embodiment, approximately 70% or more of the N-attached oligosaccharides is present on a single LAL molecule of the invention and contains terminal N-acetylglucosamine. In one embodiment, approximately 60% or more of the N-attached oligosaccharides is present on a single LAL molecule of the invention and contains terminal N-acetylglucosamine. In another embodiment, about 50% or more of the N-attached oligosaccharides is present on a single LAL molecule of the invention and contains terminal N-acetylglucosamine.

In one embodiment, all N-attached structural types of oligosaccharides present on the LAL molecules of the invention contain terminal N-acetylglucosamine. In another embodiment, about 90% or more of the N-attached oligosaccharide structural types are present on the LAL molecules of the invention and contain terminal N-acetylglucosamine. For example, if 20 types of oligosaccharide structures are present, 18 or more structural types do not contain terminal N-acetylglucosamine. In another embodiment, about 80% or more of the N-attached oligosaccharide structural types are present on the LAL molecules of the invention and contain terminal N-acetylglucosamine. In another embodiment, about 70% or more of the N-attached oligosaccharide structural types are present on the LAL molecules of the invention and contain terminal N-acetylglucosamine. In another embodiment, about 60% or more of the N-attached oligosaccharide structural types are present on the LAL molecules of the invention and contain terminal N-acetylglucosamine. In another embodiment, about 50% or more of the N-attached oligosaccharide structural types are present on the LAL molecules of the invention and contain terminal N-acetylglucosamine.

In another aspect of the invention, the compositions described herein include human LAL, with a significant percentage of human LAL not containing a fucose molecule in any of its N-glycan structures. In one embodiment, the invention is 50% or less, 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, or essentially no LAL contained in the composition does not contain a fucose molecule in any of its N-glycan structures.

In one embodiment of the invention, fucose is not substantially present in the N-linked oligosaccharide structures of the LCL obtained according to the invention. In another embodiment, approximately 95% or more of the N-attached oligosaccharides are present on a single LAL molecule of the invention without fucose. In another embodiment, approximately 90% or more of the N-linked oligosaccharides are present on a single LAL molecule of the invention without fucose. In another embodiment, approximately 85% or more of the N-attached oligosaccharides are present on a single LAL molecule of the invention without fucose. In another embodiment, about 80% or more of the N-attached oligosaccharides are present on a single LAL molecule of the invention without fucose. In another embodiment, about 70% or more of the N-attached oligosaccharides are present on a single LAL molecule of the invention without fucose. In another embodiment, about 60% or more of the N-attached oligosaccharides are present on a single LAL molecule of the invention without fucose. In another embodiment, about 50% or more of the N-attached oligosaccharides are present on the LCL of the invention without fucose.

In one embodiment of the invention, substantially none of the types of N-attached to the LAL molecules of the oligosaccharide structures of the invention contains fucose. In another embodiment, approximately 95% or more of the N-attached oligosaccharides are present on a single LAL molecule of the invention without fucose. For example, if 20 types of oligosaccharide structures are present, 19 or more structural types do not contain fucose. In another embodiment, approximately 90% or more of the N-linked oligosaccharides are present on a single LAL molecule of the invention without fucose. In another embodiment, approximately 85% or more of the N-attached oligosaccharides are present on a single LAL molecule of the invention without fucose. In another embodiment, about 80% or more of the N-attached oligosaccharides are present on a single LAL molecule of the invention without fucose. In another embodiment, about 70% or more of the N-attached oligosaccharides are present on a single LAL molecule of the invention without fucose.

As indicated above, certain monosaccharides are abundantly present in the LCL molecules prepared according to the present invention. Common monosaccharide species analyzed include fucose, N-acetylgalactosamine, N-acetylglucosamine, galactose, glucose, mannose, mannose-6-phosphate, N-acetyl neuraminic acid and N-glycolyl neuraminic acid. Fucose may be present from about 0% to about 1% of the total monosaccharide composition. N-acetylgalactosamine may be present from about 0% to about 1% of the total monosaccharide composition. N-acetylglucosamine may be present from about 35% to about 50% of the total monosaccharide composition. Galactose may be present in about 1-10% of the total monosaccharide composition. Glucose is present in 0% of the total monosaccharide composition. Mannose is present from about 32% to about 50% of the total monosaccharide composition. Mannose-6-phosphate is present from about 1% to about 11% of the total monosaccharide composition.

In one embodiment of the invention, LAL is prepared according to the present invention, and such LAL does not contain xylose. Furthermore, in view of the fact that the LAL obtained according to the invention is substantially free of N-acetylgalactosamine (GalNac), one aspect of the invention includes an LL composition that does not contain O-linked glycosylation.

LKL has 6 potential sites in its amino acid sequence for N-linked glycosylation, for example, Asn ³⁶ , Asn ⁷² , Asn ¹⁰¹ , Asn ¹⁶¹ , Asn ²⁷³ and Asn ³²¹ with the sequence SEQ ID NO: 1. Five of them, namely Asn ³⁶ , Asn ^101, Asn ¹⁶¹ , Asn ^273, and Asn ³²¹ , are glycosylated, while Asn ⁷² can be aglycosylated or essentially aglycosylated (essentially aglycosylated means that several Asn ⁷² are glycosylated in a mixture of LCL molecules compared to any Asn ³⁶ , Asn ¹⁰¹ Asn ¹⁶¹ , Asn ²⁷³ and Asn ³²¹ ) (see Fig. 17). Accordingly, one aspect of the invention describes a LKL composition that is aglycosylated and / or substantially aglycosylated at Asn ⁷² . Glycosylated Asn ⁷² LAL is also included within the scope of this invention. The Asn positions described in the text of this application are based on the amino acid sequence of the LCL of SEQ ID NO: 1. It will be apparent to those skilled in the art that the Asn numbering (i.e., the position of asparagine) can vary depending on the individual LCL molecule and can be easily defined in other LCL molecules, for example those whose sequences are represented by SEQ ID NO: 2, 3, 4 and 19.

The LCL molecules obtained according to this invention contain N-glycan structures, including a mixture of bi-, tri- and tetraantennary structures with N-acetylglucosamine, mannose and mannose-6-phosphate (M6P) as the main sugars (Fig. 16 and 17) . According to one aspect of the invention, M6P-mediated N-glycans are located on at least Asn ¹⁰¹ , Asn ¹⁶¹ and Asn ²⁷³ . Thus, one embodiment of the invention includes a composition of LCL with M6P-modified N-glycans located on any Asn ¹⁰¹ , Asn ¹⁶¹ or Asn ²⁷³ . In yet another embodiment, an LCL composition is described having M6P-modified N-glycans located on Asn ²⁷³ . In another embodiment of the present invention, an LCL composition with monophosphorylated N-glycans (M6P) located on any Asn ¹⁰¹ , Asn ¹⁶¹ or Asn ²⁷³ is described. In yet another embodiment, an LCL composition is described having monophosphorylated N-glycans located on Asn ¹⁶¹ and Asn ²⁷³ . In yet another embodiment, the invention provides an LKL composition having monophosphorylated N-glycans located on Asn ¹⁰¹ and Asn ²⁷³ . In one specific embodiment of the invention, the LCL obtained according to this invention may comprise bisphosphorylated mannose (bis-M6P) at position Asn ¹⁰¹ .

The LCL molecules obtained according to this invention contain reduced levels of galactose (for example, "Gal"). One aspect of the present invention includes an LAL composition with terminal galactose in any one of Asn ³⁶ , Asn ¹⁶¹ and Asn ³²¹ . In yet another embodiment, an LCL composition is described having terminal galactose located on Asn ³⁶ and Asn ¹⁶¹ . In yet another embodiment of the invention discloses a composition LKL having terminal galactose is disposed at Asn ¹⁶¹ and Asn ^321. In yet another embodiment, an LCL composition is described having terminal galactose located on Asn ³⁶ and Asn ³²¹ . In yet another embodiment, an LCL composition is described having terminal galactose located on Asn ³⁶ , Asn ¹⁶¹ and Asn ³²¹ . In yet another embodiment of the invention, an LCL composition not containing terminal galactose is described.

In LCL, various types of N-glycans are located in different N-linked glycosylation sites. N-glycosylated structures include a mixture of bi-, tri- and tetraantennary structures with N-acetylglucosamine, mannose and mannose-6-phosphate (M6P) as basic sugars. In particular, in one embodiment, the LAL contains an N-glycan structure selected from GlcNAc4Man3 GlcNAc2 or GallGlcNAc4Man3GlcNAc2 at the first N-linked glycosylation site (e.g., Asn ³⁶ with the sequence SEQ ID NO: l). In another embodiment, the LAL does not contain glycosylation or is substantially aglycosylated at the second N-linked glycosylation site (e.g., Asn ⁷² with the sequence SEQ ID NO: l). In yet another embodiment, the LAL contains Phos2Man7GlcNAc2 at the third N-linked glycosylation site (e.g., Asn ¹⁰¹ with the sequence SEQ ID NO: l). In yet another embodiment LKL comprises N-glycan structures selected from PhoslMan6GlcNAc2, GlcNAclPhoslMan6GlcNAc2, Man3GlcNAc2, GlcNAc2Man3GlcNAc2, GlcNAc3Man3GlcNAc2, GlcNAc4Man3GlcNAc2 or Gall GlcNAc4Man3GlcNAc2, in the fourth N-attached glycosylation site (e.g., Asn ¹⁶¹ with the sequence SEQ ID NO: l) . In yet another embodiment, the LAL contains an N-glycan structure selected from Man7GlcNAc2, Man8GlcNAc2, Man9GlcNAc2, PhoslMan8GlcNAc2 or PhoslMan9GlcNAc2, in the fifth N-linked glycosylation site (e.g., Asn ²⁷³ with l sequence Asn ²⁷³ NO: l). In yet another embodiment LKL invention comprises N-glycan structure selected from the GlcNAc2Man3GlcNAc2, GlcNAc3Man3GlcNAc2, GlcNAc4Man3GlcNAc2, GallGlcNAc4Man3GlcNAc2, GlcNAc5Man3GlcNAc2, GallGlcNAc5Man3GlcNAc2, GlcNAc6Man3GlcNAc2 or GallGlcNAc6Man3GlcNAc2, in the sixth N-attached glycosylation site (e.g., Asn ³²¹ with the sequence SEQ ID NO: l )

According to certain aspects of the invention, LKL compositions include LKL glycosylated at positions Asn ³⁶ , Asn ⁷² , Asn ^101, Asn ¹⁶¹ , Asn ²⁷³ and Asn ^{321 of the} sequence SEQ ID NO: l (or the corresponding asparagine residues in the sequences SEQ ID NO: 2, 3, 4 and 19) in one N-glycan in the indicated position of Asn, as defined below:

a) at position Asn ³⁶ , GlcNAc4Man3GlcNAc2 or

GallGlcNAc4Man3GlcNAc2;

b) at position Asn ⁷² , lack of glycosylation;

c) at position Asn ¹⁰¹ , Phos2Man7GlcNAc2;

d) at position Asn ¹⁶¹ , PhoslMan6GlcNAc2,

GlcNAc 1 Phos 1 Man6GlcNAc2;

Man3GlcNAc2;

GlcNAc2Man3GlcNAc2;

GlcNAc3Man3GlcNAc2;

GlcNAc4Man3GlcNAc2 or

GallGlcNAc4Man3GlcNAc2;

e) at position Asn ²⁷³ , Man7GlcNAc2,

Man8GlcNAc2,

Man9GlcNAc2,

PhoslMan8GlcNAc2, or

PhoslMan9GlcNAc2; and

f) at position Asn ³²¹ , GlcNAc2Man3GlcNAc2,

GlcNAc3Man3GlcNAc2,

GlcNAc4Man3GlcNAc2,

GallGlcNAc4Man3GlcNAc2,

GlcNAc5Man3GlcNAc2,

GallGlcNAc5Man3GlcNAc2,

GlcNAc6Man3GlcNAc2, or

GallGlcNAc6Man3GlcNAc2,

where Man = mannose,

GlcNAc = N-Acetylglucosamine,

Phos = phosphate and

Gal = galactose.

In one embodiment, GallGlcNAc4Man3GlcNAc2 may be present as a glycan component at any of the positions of Asn ³⁶ , Asn ¹⁶¹ or Asn ³²¹ in the LCL obtained according to this invention. In one specific embodiment, GallGlcNAc4Man3GlcNAc2 may be present as a glycan component of Asn ³⁶ , Asn ¹⁶¹ and Asn ³²¹ .

In the LCL of this invention, Asn ¹⁰¹ and Asn ²⁷³ contain an increased amount of mannose from about 6 to about 10 mannose molecules (MAN6-MAN10, as described herein) as a main component. Accordingly, one aspect of the present invention includes a high mannose LKL composition at position Asn ¹⁰¹ or Asn ²⁷³ . In another embodiment, the invention LKL composition of the invention may include N-glycan structure having at least six mannose at positions Asn ¹⁰¹ or Asn ^273. In another embodiment, the LKL composition comprises N-glycan having 7, 8 or 9 mannose at positions Asn ¹⁰¹ and Asn ²⁷³ . In yet another embodiment, the LKL composition comprises 7, 8, or 9 mannose at positions Asn ¹⁰¹ and / or Asn ²⁷³ and at least one mannose is phosphorylated.

It should be understood that the glycosylation sites and the number of these sites associated with the Asn described above are based on the amino acid sequence of LKL with SEQ ID NO: 1, and the glycosylation profiles described above in the context of SEQ ID NO: 1 are also applicable to LKL molecules with sequences SEQ ID NO: 2, 3, 4, and 19, however, the numbering of the corresponding Asn may vary depending on the LCL molecule. For example, Asn ³⁶ in SEQ ID NO: 1 corresponds to Asn ¹⁵ in SEQ ID NO: 2, Asn ¹³ in SEQ ID NO: 3, Asn ¹⁰ in SEQ ID NO: 4, and Asn ⁹ in SEQ ID NO: 19. Asn ⁷² in SEQ ID NO: 1 corresponds to Asn ⁵¹ in SEQ ID NO: 2, Asn ⁴⁹ in SEQ ID NO: 3, Asn ⁴⁶ in SEQ ID NO: 4, and Asn ⁴⁵ in SEQ ID NO: 19. Asn ¹⁰¹ in SEQ ID NO: 1 corresponds to Asn ⁸⁰ in SEQ ID NO: 2, Asn ⁷⁸ in SEQ ID NO: 3, Asn ⁷⁵ in SEQ ID NO: 4, and Asn ⁷⁴ in SEQ ID NO: 19. Asn ¹⁶¹ in SEQ ID NO: 1 corresponds to Asn ¹⁴⁰ in SEQ ID NO: 2, Asn ¹³⁸ in SEQ ID NO: 3, Asn ¹³⁵ in SEQ ID NO: 4, and Asn ¹³⁴ in SEQ ID NO: 19. Asn ²⁷³ in SEQ ID NO: 1 corresponds to Asn ²⁵² in SEQ ID NO: 2, Asn ²⁵⁰ in SEQ ID NO: 3, Asn ²⁴⁷ in SEQ ID NO: 4, and Asn ²⁴⁶ in SEQ ID NO: 19. Asn ³²¹ in SEQ ID NO: 1 corresponds to Asn ³⁰⁰ in SEQ ID NO: 2, Asn ²⁹⁸ in SEQ ID NO: 3, Asn ²⁹⁵ in SEQ ID NO: 4, and Asn ²⁹⁴ in SEQ ID NO: 19.

For example, in one embodiment, the LCL is in an N-attached form glycosylated in at least one position selected from the group consisting of Asn ¹⁵ , Asn ⁵¹ , Asn ⁸⁰ , Asn ¹⁴⁰ , Asn ^252, and Asn ^{300 of} the SEQ sequence ID NO: 2. In another embodiment, the LCL is provided in an N-attached form, glycosylated at the position of Asn ¹⁵ , Asn ⁸⁰ , Asn ¹⁴⁰ , Asn ²⁵² and Asn ^{300 of the} sequence SEQ ID NO: 2. In another embodiment, the N-glycan structures of SEQ ID NO: 2 do not contain xylose, while less than 15%, 10%, 5%, or 1% of the N-glycan structures contain sialic acid; less than 50%, 40%, 30%, 20%, 10%, 5% or 1% of N-glycan structures contain fucose; and at least 30%, 50%, 60%, 70%, 80%, 90% and 95% of N-glycan structures contain phosphorylated mannose (M6P).

In one embodiment, the LCL is in an N-attached form glycosylated in at least one position selected from the group consisting of Asn ¹³ , Asn ⁴⁹ , Asn ⁷⁸ , Asn ¹³⁸ , Asn ^250, and Asn ^{298 of the} sequence SEQ ID NO : 3. In another embodiment, the LCL is provided in an N-attached form glycosylated at the position of Asn ¹³ , Asn ⁷⁸ , Asn ¹³⁸ , Asn ²⁵⁰ and Asn ^{298 of the} sequence SEQ ID NO: 3. In another embodiment, the N-glycan structures of SEQ ID NO: 3 do not contain xylose, while less than 15%, 10%, 5%, or 1% of the N-glycan structures contain sialic acid; less than 50%, 40%, 30%, 20%, 10%, 5% or 1% of N-glycan structures contain fucose; and at least 30%, 50%, 60%, 70%, 80%, 90% and 95% of N-glycan structures contain phosphorylated mannose (M6P).

In one embodiment, the LCL is in an N-attached form glycosylated in at least one position selected from the group consisting of Asn ¹⁰ , Asn ⁴⁶ , Asn ⁷⁵ , Asn ¹³⁵ , Asn ²⁴⁷ and Asn ^{295 of the} sequence SEQ ID NO :four. In another embodiment, the LCL is provided in an N-attached form glycosylated at the position of Asn ¹⁰ , Asn ⁷⁵ , Asn ¹³⁵ , Asn ²⁴⁷ and Asn ^{295 of the} sequence SEQ ID NO: 4. In another embodiment, the N-glycan structures of SEQ ID NO: 4 do not contain xylose, while less than 15%, 10%, 5%, or 1% of the N-glycan structures contain sialic acid; less than 50%, 40%, 30%, 20%, 10%, 5% or 1% of N-glycan structures contain fucose; and at least 30%, 50%, 60%, 70%, 80%, 90% and 95% of N-glycan structures contain phosphorylated mannose (M6P).

In one embodiment, the LCL is in an N-attached form glycosylated in at least one position selected from the group consisting of Asn ⁹ , Asn ⁴⁵ , Asn ⁷⁴ , Asn ¹³⁴ , Asn ^246, and Asn ^{294 of the} sequence SEQ ID NO :19. In another embodiment, the LCL is provided in an N-attached form glycosylated at the position of Asn ⁹ , Asn ⁷⁴ , Asn ¹³⁴ , Asn ²⁴⁶ and Asn ^{294 of the} sequence SEQ ID NO: 19. In another embodiment, the N-glycan structures of SEQ ID NO: 4 do not contain xylose, while less than 15%, 10%, 5%, or 1% of the N-glycan structures contain sialic acid; less than 50%, 40%, 30%, 20%, 10%, 5% or 1% of N-glycan structures contain fucose; and at least 30%, 50%, 60%, 70%, 80%, 90% and 95% of N-glycan structures contain phosphorylated mannose (M6P).

The composition of this invention can be obtained using a variety of methods, including the use of transgenic birds, transgenic fish, transgenic mammals, for example, transgenic goats or transgenic plants such as tobacco and duckweed (Lemna minor), as well as certain types of cell structures.

The invention also provides compositions comprising pegylated LCL. The LKL enzyme described herein may be pegylated as described, for example, in US Pat. 20070092486 dated April 26, 2007, the contents of which are hereby incorporated by reference in their entirety.

In one embodiment of the invention, the presented glycosylation scheme was obtained by specialized expression systems, for example, from avian oviduct cells, for example, tubular gland cells. For example, it has been shown that the glycosylation patterns described herein are present in the enzymes of lysosomal storage diseases produced in the cells of avian oviducts, such as chicken, according to this invention.

Proteins obtained in accordance with the invention can be purified from egg white using any suitable protein purification procedure known to one skilled in the art. For example, human LKL (hLKL) obtained from transgenic birds according to the present invention can be purified from egg protein using protein purification methods known to those skilled in the art. An example of a protocol for purifying LCL from egg white is described in the Examples.

The invention includes eggs and egg whites, as well as birds (e.g., chickens, turkeys, and quails) that lay eggs and produce egg white containing lysosomal acid lipase molecules of the invention, comprising one or more of the glycosylated structures described herein.

EXPRESSION OF LCL AT BIRDS

This invention describes vectors and methods for the stable incorporation of exogenous nucleic acid sequences into the avian genome to express desired proteins, for example, those that are improved (for example, given increased efficiency) by adding mannose-6-phosphate, such as lysosomal enzymes, including , without limitation, lysosomal acid lipase (LCL) and other proteins, such as those described in the text of this application. In particular, transgenic birds are obtained that express exogenous sequences in their oviducts with the deposition of exogenous proteins, such as pharmaceutical proteins, in the eggs. Bird eggs containing such exogenous proteins are also described in this invention. Also described are the latest forms of LCL that are efficiently expressed in the oviduct of transgenic birds and deposited in bird eggs.

One aspect of the present invention describes compositions containing LAL, i.e. LCL molecules obtained according to the invention. In a separate embodiment of the invention, the LAL is purified or isolated. For example, LAL was purified from the contents of the hard shell of eggs laid by a transgenic bird. In one particular embodiment of the invention, LAL is human LAL. In one embodiment of the invention, the LAL of the invention has a glycosylation pattern derived from LAL produced in the cells of a bird's oviduct. For example, the compositions may contain a mixture of LCL molecules obtained from birds, for example, chickens, in accordance with the invention, and isolated from egg white. In one embodiment of the invention, compositions containing LAL are pharmaceutical compositions.

In one aspect, the invention provides compositions containing isolated LAL molecules, for example, human LAL molecules, wherein LAL is obtained from birds that contain a transgene encoding LAL. In one embodiment, LCL is obtained in an oviduct cell (eg, tubular cell) of a transgenic bird (eg, transgenic chicken), and LCL is isolated from egg protein of a transgenic bird. In one embodiment, LAL is glycosylated in an oviduct cell (eg, tubular cell) of a bird, such as a chicken.

In another aspect of the invention, methods for producing exogenous proteins, such as enzymes of lysosomal storage disease, for example, LAL, in specific tissues of birds are described. Such exogenous proteins can be expressed in the oviduct, blood cells and / or other cells and tissues of the bird. In one embodiment of the invention, transgenes are introduced into embryonic blastoderm cells, for example, in stage X, to obtain a transgenic bird, thus, the desired protein is expressed in the tubular gland cells of the oviduct magnum, secreted into the duct, and deposited in the egg protein of the hard shell of eggs. The transgenic bird thus obtained can carry the transgene in the cells of the germ line, ensuring the transmission of the exogenous transgene to its offspring according to the rules of Mendel.

This invention encompasses methods for producing an exogenous protein, such as LAL, in the oviduct of a bird. The methods may include the first step of providing a vector comprising a coding sequence and a promoter operably linked to the coding sequence, so the promoter can affect the expression of nucleic acid in the bird's oviduct. Transgenic cells and / or tissues can be obtained by introducing the vector into the embryonic blastoderm cells of the bird that have been freshly isolated, are in the culture or in the embryo, so the sequence of the vector is incorporated into the bird genome. A mature transgenic bird expressing an exogenous protein, such as LAL, in its oviduct can be obtained from transgenic cells and / or tissue.

In one aspect of the invention, the production of a transgenic bird is accompanied by transduction of embryonic blastoderm cells using replication-defective or replication-competent retroviral particles carrying a transgene between the 5 ′ and 3 ′ LTR retroviral vector. For example, a bird leukemia virus (ALV) retroviral vector or a mouse leukemia virus (MLV) retroviral vector can be used, which include a modified pNLB plasmid containing an exogenous gene inserted below a segment of a promoter site. A copy of the RNA of the modified retroviral vector packaged in viral particles can be used to infect embryonic blastoderms developing in transgenic birds.

Another aspect of the present invention describes a vector that includes a coding sequence and a promoter in a functional and positional ratio, so that a coding sequence is expressed in the oviduct of a bird. Such vectors include, but are not limited to, avian leukemia virus (ALV) retroviral vector, mouse leukemia virus (MLV) retroviral vector, and lentivirus vector. In addition, the vector can be represented by a nucleic acid sequence that includes the LTR of the avian leukemia virus retroviral vector (ALV), mouse leukemia virus retroviral vector (MLV) or lentivirus vector. The promoter is effective enough to control the expression of the coding sequence in the oviduct of the bird. The coding sequence encodes an exogenous protein that is deposited in the egg protein of the hard shell of an egg. Thus, the coding sequence encodes exogenous proteins that can be obtained from transgenic birds, such as lysosomal acid lipase obtained from transgenic birds (TPD LCL).

In one embodiment, the vectors used in the methods of the invention comprise a promoter that is particularly suitable for the expression of exogenous proteins in birds and their eggs. Thus, the expression of an exogenous coding sequence can occur in the oviduct and blood of a transgenic bird and in the egg protein of a bird egg. Promoters include, but are not limited to, the cytomegalovirus (CMV) promoter, the raush sarcoma virus (RSV) promoter, the P-actin promoter (e.g., the chicken P-actin promoter), the mouse leukemia virus (MLV) promoter, the mouse breast tumor virus promoter (MMTV), egg albumin promoter, lysozyme promoter, conalbumin promoter, ovomucoid promoter, ovomucin promoter and ovotransferrin promoter. In some cases, the promoter may be represented by a segment of at least one portion of the promoter, such as a segment of egg albumin, lysozyme, conalbumin, ovomucoid, ovomucin, and the ovotransferrin promoter region. In one embodiment, the promoter is a combination or fusion of one or more promoters or a fusion of a portion of one or more promoters, such as egg albumin, lysozyme, conalbumin, ovomucoid, ovomucin and ovotransferrin promoters.

In one embodiment of the invention, the vector comprises a coding sequence for a signal peptide that is operably linked to a coding sequence, so that, after being translated into a cell, the signal peptide directs the secretion of an exogenous protein expressed by a vector, such as human LAL, into the egg white.

One aspect of the present invention describes coding sequences for exogenous proteins obtained as described herein, and such a coding sequence is represented by a codon optimized for expression in birds, such as chickens. Codon optimization can be determined by using a codon to express at least one and, preferably, more than one protein in a bird cell (eg, chicken cell). For example, the use of a codon can be determined based on nucleic acid sequences encoding proteins of egg albumin, lysozyme, ovomucin and chicken ovotransferrin. For example, the coding DNA sequence for an exogenous protein can be represented by an optimized codon using the BACKTRANSLATE® program from the Wisconsin Package, version 9.1 (Genetics Computer Group, Inc., Madison, WI), which provides a codon table for egg albumin, lysozyme, ovomucoid and chicken ovotransferrin (Gallus gallus).

One important aspect of the present invention describes the hard shell of bird eggs (e.g., hard-shell chicken eggs) that contain an exogenous peptide or protein, including, but not limited to, human LAL. An exogenous peptide or protein, such as human LAL, can be encoded by a transgenic bird transgene. Very often, an exogenous protein or peptide (e.g., LAL) is glycosylated. Protein may be present in any suitable amount. In one embodiment, the protein is present in an amount in the range of from about 0.01 μg per hard-shell egg to about 1 g per hard-shell egg. In another embodiment, the protein is present in an amount in the range of from about 1 μg per hard-shell egg to about 1 g per hard-shell egg. For example, protein may be present in an amount ranging from about 10 μg per hard-shell egg to about 1 g per hard-shell egg (for example, a range from about 10 μg per hard-shell egg to about 400 mg per hard-shell egg) .

In one embodiment, an exogenous protein of the invention is present in the egg white of an egg. In one embodiment, the protein is present in an amount in the range of from about 1 ng per ml of egg white to about 0.2 g per ml of egg white. For example, protein may be present in an amount in the range of about 0.1 μg per ml of egg white to about 0.2 g per ml of egg white (for example, protein may be present in an amount in the range of about 1 μg per ml of egg white to about 100 mg per ml of egg white). In one embodiment, the protein is present in an amount in the range of from about 1 μg per ml of egg white to about 50 mg per ml of egg white. For example, protein can be present in an amount in the range of about 1 μg per ml of egg white to about 10 mg per ml of egg white (for example, protein can be present in an amount in the range of about 1 μg per ml of egg white to about 1 mg per ml of egg squirrel). In one embodiment, the protein is present in an amount of more than 0.1 μg per ml of egg white. In one embodiment, the protein is present in an amount of more than 0.5 μg per ml of egg white. In one embodiment, the protein is present in an amount of more than 1 μg per ml of egg white. In one embodiment, the protein is present in an amount of more than 1.5 μg per ml of egg white.

The birds of the invention producing the exogenous proteins described in the text of this application (for example, LCL), developed from blastoderm cells into which the vector was introduced, are the G0 generation and can be referred to as the "founders". Founding birds are usually chimeras for each inserted transgene. Thus, some G0 transgenic bird cells contain transgene (s). The G0 generation is usually hemizygous for the transgene (s). Generation G0 can be propagated to non-transgenic animals to produce transgenic progeny G1, which are also hemizygous for the transgene and contain transgene (s) in essentially all of their cells. Hemizygous progeny of G1 can be crossed with non-transgenic animals to obtain hemizygous progeny of G2 or can be crossed among themselves to obtain offspring of G2 homozygous for the transgene. Essentially all bird cells positive for a transgene and derived from G1 progeny contain transgene (s). In one embodiment, the hemizygous progeny of G2 from the same line can be crossed to produce a homozygous progeny of G3 for a transgene. In one embodiment of the invention, the hemizygous animals G0 or G1, for example, mate to produce homozygous offspring G1 containing two copies of the transgene (s) in each animal cell. These are just a few examples of specific crossbreeding methods, and this invention covers the use of any suitable crossbreeding method known to those skilled in the art.

In one embodiment, the invention describes isolated LCL. Such LCL contained in the composition may be represented by isolated LCL. For example, LAL can be isolated from egg white. Isolated LKL can be represented by LKL molecules having various glycosylated structures among LKL molecules.

By using the methods of this invention, transgenes can be introduced into avian embryonic blastoderm cells to produce transgenic hens, transgenic turkeys, transgenic quail and other species of birds carrying the transgene in the genomic material of their cell line to produce the proteins of the invention. Blastodermal cells are typically represented by cells in stage VII-XII or their equivalents, and in one embodiment of the invention are in stage X.

The text of this application describes some vectors used in the methods of this invention. In one embodiment of the invention, the coding sequence and promoter of the vector are located between the 5 ′ and 3 ′ LTR before being introduced into the blastoderm cells. In one embodiment, the vector is retroviral, and the coding sequence and promoter are located between the 5 ′ and 3 ′ LTR of the retroviral vector. In one embodiment, the LTR or retroviral vector is derived from avian leukemia virus (ALV), mouse leukemia virus (MLV), or lentivirus.

In one embodiment of the invention, vectors are used to transfect blastoderm cells and obtain stable integration into the avian genome containing a coding sequence and a promoter in a functional and positional relationship for expressing the coding sequence in the tubular gland cells of avian oviduct magnum, while an exogenous protein, such as a lysosomal enzyme (e.g., LAL) is deposited in the egg white of an egg with a hard shell.

The promoter in some cases can be represented by a segment of the egg albumin promoter region that is large enough to control the expression of the coding sequence in the cells of the tubular gland. The branching of the egg albumin promoter and / or the condensation of important regulatory elements of the egg albumin promoter while preserving the sequences necessary for expression of the oviduct magnum in the tubular gland cells, while being small enough for easy introduction into the vectors, is included in the scope of the invention. In one embodiment of the invention, a segment segment of an egg albumin promoter may be used. Such a segment includes a 5'-flanking portion of the egg albumin gene.

The promoter can also be represented by a promoter that is more, but not completely, specific for magnum, such as the lysozyme promoter. The promoter may also be represented by the promoter of the mouse breast tumor virus (MMTV). Alternatively, the promoter may be represented by a constitutive promoter (e.g., a cytomegalovirus (CMV) promoter, a raush sarcoma virus (RSV) promoter, a mouse leukemia virus (MLV) promoter, etc.). In one embodiment, the promoter is a cytomegalovirus (CMV) promoter, an MDOT promoter, a rash sarcoma virus (RSV) promoter, a mouse leukemia virus (MLV) promoter, a mouse breast tumor virus (MMTV) promoter, an egg albumin promoter, a lysozyme promoter, a conalbumin promoter, an ovomukoid promoter, an ovomucin promoter and / or an ovotransferrin promoter. In some cases, the promoter may be represented by a segment of at least one portion of the promoter, such as a segment of egg albumin, lysozyme, conalbumin, ovomucoid, ovomucin, and the ovotransferrin promoter region.

In a method for transfection of blastoderm cells, a packaged vector based on a retrovirus is used to deliver the vector to embryonic blastoderm cells, thus the vector is integrated into the avian genome.

Retroviruses used to randomly integrate a transgene into the bird genome are replication-incapable avian leukemia virus (ALV), replication-incapable mouse leukemia virus (MLV) or lentivirus. In one embodiment, the pNLB vector is modified by inserting a portion of the egg albumin promoter and one or more exogenous genes between the 5 'and 3' long terminal repeats (LTR) of the retrovirus genome. The invention provides that any coding sequence downward from a promoter active in tubular gland cells can be expressed in tubular gland cells. For example, the egg albumin promoter can be expressed in tubular gland cells of the magnum oviduct because the egg albumin promoter directs the expression of egg albumin protein and is active in the tubular gland cells of the oviduct.

Any vectors described herein may also in some cases include a coding sequence that encodes a signal peptide directing the secretion of a protein expressed by the coding sequence of the vector from tubular cells of the oviduct. This aspect effectively extends the spectrum of exogenous proteins that can be deposited in bird eggs using the methods described herein. Considering that exogenous proteins are not secreted otherwise, the vector containing the coding sequence is modified to include a DNA sequence containing approximately 60 bp encoding the signal peptide of 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 includes the use of elements of the ribosome internal landing sites (IRES) in any of the vectors of the present invention to allow translation of two or more proteins from bicistronic or polycistronic mRNA. IRES units are fused to the 5'-ends of one or more additional coding sequences, which are then inserted into the vectors at the end of the original coding sequence, thus the coding sequences are separated from each other by IRES.

In one embodiment of the invention, using IRES, post-translational modification of the product is facilitated because one coding sequence can encode an enzyme capable of modifying the product of a different coding sequence. For example, the first coding sequence can encode collagen, which will be hydroxylated and activated by the enzyme encoded by the second coding sequence, while IRES is used as is customary in science.

In another aspect, the coding sequences of vectors used in any method of the invention are described with 3 ′ untranslated regions (3 ′ UTR) to confer stability to the resulting RNA. When 3 ′ UTR is added to the retroviral vector, the orientation of the promoter, X gene, and 3 ′ UTR in the vector must be reversed so that the addition of 3 ′ UTR does not affect the transcription of full length genomic RNA. In one embodiment, the 3 'UTR can be derived from egg albumin or lysozyme genes, or any 3' UTR is functional in magnum cells, i.e. plot SV40.

In one embodiment, a constitutive promoter is used to express the coding sequence of the transgene in birds. In this case, expression is not limited to magnum; expression also occurs in other tissues of the bird (e.g., blood). The use of such a transgene, which includes a constitutive promoter and a coding sequence, is particularly convenient for influencing or directing protein expression in the oviduct and subsequent secretion of the protein in the egg.

Converting particles (i.e., transfected particles) are prepared for the vector and used to determine the appropriate concentration that can be used to inject the embryos. Holes in the eggs of the birds are made according to the Speksnijder method (US Pat. No. 5,897,998, the contents of which are incorporated herein in their entirety by reference), and transfected particles are introduced into the eggs. The eggs hatch within 21 days after injection, and male birds are selected for breeding. For screening G0 cocks containing a transgene in their semen, DNA is extracted from the cocks sperm samples. G0 roosters with the highest transgene levels in semen samples are used for crossbreeding with non-transgenic chickens by artificial insemination. Blood DNA samples are screened for the presence of a transgene. Transgenic rooster serum is tested for the presence of exogenous protein. In the case of confirmation of exogenous protein, the sperm of transgenic cocks is used for artificial insemination of non-transgenic chickens. Subsequently, a certain percentage of offspring contains a transgene (for example, more than 50%). In the presence of exogenous protein in the eggs obtained according to this invention, the protein can be isolated. Protein can also be tested for biological activity.

The methods of the invention, providing an exogenous protein in the oviduct of birds and obtaining eggs containing an exogenous protein, include the additional step of providing a suitable vector and introducing the vector into embryonic blastoderm cells so that the vector is integrated into the bird genome. The next step involves obtaining a mature transgenic male bird from transgenic blastoderm cells obtained in the previous steps. Mature transgenic birds can be obtained from blastodermal embryo cells that have been transfected or have been introduced directly into the embryo. The resulting embryo develops and the chicken is allowed to grow.

A transgenic bird derived from blastoderm cells is called the founder. Some founders will carry a transgene in the cells of the tubular gland of their oviduct. Such birds will express an exogenous protein encoded by a transgene in the oviduct. Exogenous protein, in addition to the oviduct, can also be expressed in other tissues (e.g., blood). If the exogenous protein contains the corresponding signal sequence (s), it will be secreted in the duct of the oviduct and in the egg protein.

Some founders are the founders of the germ line. The founder of the germ line is represented by the founder carrying the transgene in the genetic material, the tissues of the germ line, and can also carry the transgene in the tubular gland cells of the oviduct magnum that express the exogenous protein. Thus, according to the invention, the transgenic bird can have tubular gland cells expressing an exogenous protein, and the progeny of the transgenic bird can also have tubular gland cells of the oviduct magnum expressing an exogenous protein. On the other hand, the offspring expresses a phenotype determined by the expression of the exogenous gene in the specific tissue (s) of the bird. In one embodiment, the transgenic bird is chicken or turkey.

PHARMACEUTICAL COMPOSITIONS AND METHODS OF TREATMENT

Having the possibility of use in therapy, therapeutic proteins obtained in accordance with this invention can be introduced in crude form, however, mainly their introduction as part of a therapeutic composition. Thus, pharmaceutical compositions are further described comprising glycosylated therapeutic proteins obtained from birds, such as LAL, or their pharmaceutically acceptable derivatives together with one or more pharmaceutically acceptable carriers, and, in some cases, other therapeutic and / or prophylactic ingredients and methods the introduction of such pharmaceutical compositions. The carrier (s) must be “acceptable” in the sense of being compatible with the other ingredients of the composition and not be detrimental to the recipient. Methods of treating a patient (for example, the amount of pharmaceutical protein administered, frequency of administration and duration of treatment period) using the pharmaceutical compositions of the invention can be determined using standard methods known to those skilled in the art.

Compositions comprising carriers, including composite molecules, have been developed by conventional methods (see, for example, Remington's Pharmaceutical Sciences, 14th Ed., Mack Publishing Co., Easton, Pa.), The contents of which are incorporated herein in their entireties by reference. The carrier may include a diluent. In one embodiment, the pharmaceutical carrier may be a liquid, and the recombinant human LAL may be in the form of a solution. The pharmaceutical carrier may be represented by wax, fat, or alcohol. In one embodiment of the invention, the wax or fat based carrier is free of ether. In another embodiment, the pharmaceutically acceptable carrier may be a solid in the form of a powder, lyophilized powder, or tablet. In one embodiment, the carrier may include a liposome or microcapsule.

Pharmaceutical compositions include compositions suitable for administration by injection, including intramuscular, subcutaneous and intravenous administration. Pharmaceutical compositions include compositions suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral administration. Pharmaceutical compositions also include compositions for administration by inhalation or insufflation. Compositions, where possible, can be conveniently presented in unit dosage form and can be obtained using any method well known in pharmacy. Methods for preparing pharmaceutical compositions typically include the step of contacting the therapeutic protein with liquid carriers or finely divided solid carriers or both, and then, if necessary, molding the product into the desired composition.

Pharmaceutical compositions suitable for oral administration may conveniently be presented as discrete units, such as capsules, sachets or tablets, each of which contains a predetermined amount of active active ingredient; in the form of powder or granules; in the form of a solution; in the form of a suspension; or in the form of an emulsion. The active substance may also be presented as a bolus dose, electuary or paste. Tablets and capsules for oral administration may contain a sufficient amount of excipients, such as binders, fillers, lubricants, disintegrants or wetting agents. Tablets may also be coated according to methods known in the art. Oral liquid compositions may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or another suitable vehicle before use. Such liquid compositions may contain suitable additives, such as suspending agents, emulsifiers, non-aqueous vehicles (which may include edible oils) or preservatives.

LAL can also be presented in a composition for parenteral administration (for example, by administering, for example, a bolus injection or continuous infusion) and can be presented in dosage form in ampoules, prefilled syringes, small volume infusion containers or multi-dose containers with added preservative. Therapeutic proteins can be administered, for example, by subcutaneous injection, intramuscular injection and intravenous injection or infusion.

LAL can be presented in such forms as suspensions, solutions or emulsions in oil or liquid carriers, and may contain molding agents, such as suspending, stabilizing and / or dispersing agents. It is also contemplated that the therapeutic protein may be presented in powder form, obtained by aseptic isolation of a sterile solid or by lyophilization from solution, for reconstitution with a suitable vehicle, for example, sterile, pyrogen-free water, before use.

For intravenous infusions or injections, LAL obtained in accordance with this invention may be presented in the form of an aqueous suspension or solution. Excipients suitable for use in an intravenous infusion or injection composition may include one of the following: trisodium citrate dehydrate, citric acid, and human serum albumin.

The pharmaceutical composition may also include other suitable excipients, well known in science for other products for lysosomal storage disorders. The pH of the LAL obtained according to this invention is between about 5.6 and 6.2. Advantageously, the pH of the LCL composition is 5.9 ± 0.2.

For topical administration into the epidermis, the therapeutic proteins of the invention obtained according to the invention can be presented as ointments, creams or lotions, or as a skin patch. Ointments and creams may include, for example, a water or oil base with the addition of suitable thinning and / or gelling agents. Lotions in their composition may include an oily or aqueous base and may also contain one or more emulsifiers, stabilizers, dispersing agents, suspending agents, thinning agents or coloring agents.

Compositions suitable for topical administration in the mouth include lozenges comprising an active substance with a flavored base, usually sucrose and gum or tragacanth; lozenges comprising the active substance on an inert basis, such as gelatin and glycerin or sucrose and gum; and mouthwashes comprising the active substance in a suitable liquid carrier.

Pharmaceutical compositions suitable for rectal administration include a solid carrier, and such compositions are preferably presented as single dose suppositories. Suitable carriers include cocoa butter and other materials that are commonly used in science, and suppositories can be formed by a mixture of the active active ingredient with emollient or melting carriers, followed by mixing and molding in the molds.

Compositions suitable for vaginal administration can be presented in the form of pessaries, tampons, creams, gels, pastes, foams or spray containing, in addition to the active active substance, carriers which are known in science.

For intranasal administration, the therapeutic proteins of the invention can be used as a liquid spray or dispersible powder or in the form of drops. Drops may contain an aqueous or non-aqueous base, which also includes one or more dispersing agents, solubilizers or suspending agents. Liquid sprays are conveniently supplied in pressure packs.

For administration by inhalation, the therapeutic proteins of the invention may conveniently be presented as an insufflator, nebulizer or pressure pack or in another convenient form for delivery of an aerosol spray.
Pressurized packages may include a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dose unit can be determined by placing a valve to deliver a metered amount to the body.

For administration by inhalation or insufflation, the therapeutic proteins of the invention may be presented in the form of a dry powder composition, for example, a mixture of the powders of the compounds and a suitable powder base, such as lactose or starch. The powder composition may be presented in a unit dosage form, for example, in capsules or cartridges, or, for example, gelatin or blister packs, the powder may be administered using an inhaler or insufflator.

If necessary, the above compositions are adapted to obtain a sustained release of the active active ingredient.

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

In addition, it is contemplated that the therapeutic proteins described herein may be used in combination with other therapeutic agents. For example, the invention describes pretreatment methods using a pharmaceutically effective dose of an antihistamine to minimize or prevent anaphylactic reactions caused by infusion. For example, an antihistamine may be any pharmaceutically acceptable antihistamine (e.g., diphenhydramine), as described in this invention and is known in the art. In one embodiment, an antihistamine is administered at a dose of about 1 mg to about 10 mg per kg body weight. For example, an antihistamine may be administered at a dose of about 5 mg per kg. In one embodiment, an antihistamine is administered for a period of from about 10 minutes to about 90 minutes, for example from about 30 minutes to about 60 minutes, before administering a lysosomal acid lipase using an outpatient system connected to a vascular access port. In one embodiment, the dose of diphenhydramine effectively counteracts potential anaphylactic infusion reactions.

Immunosuppressants such as antihistamines, corticosteroids, sirolimus, voklosporin, cyclosporin, methotrexate, antibodies directed to the IL-2 receptor, antibodies to T -cellular receptor, antibodies to TNF-alpha or fusion proteins (infliximab, etanercept or adalimumab), CTLA4-Ig (for example, abatacept) or antibodies to OX-40.

The invention also provides a treatment comprising administering compositions containing LAL in combination with one or more cholesterol lowering agents (e.g., HMG-CoA reductase inhibitors). Non-limiting examples of such agents include: atorvastatin (Lipitor® and Torvast®), fluvastatin (Leskol®), lovastatin (Mevkor®, Altokor®, Altoprev®), pitavastatin (Livalo®, Pitava®), pravastatin (Pravahol®, Selectin®, Lipostat®), rosuvastatin (Crestor®) and simvastatin (Zokor®, Lipex®).

The compositions or proteins described can be used to treat various conditions. For example, there are conditions whose treatment principles are known to those skilled in the art. The present invention provides that therapeutic proteins (e.g., LAL) obtained in the avian system and containing the glycosylation pattern obtained in birds can be used to treat such conditions. Therefore, treatment is provided for conditions that are known to be treated with therapeutic proteins obtained by conventional methods by using therapeutic proteins obtained according to the invention. For example, the LAL obtained by the invention can be used to treat conditions that result from or are associated with a LAL deficiency or deficiency (the general concept of “LAL deficiency”), such as Wolman’s disease and cholesterol ester accumulation disease (BNEC). As described in the text of this application, the LKL deficiency also provides conditions in which the expression of LKL is reduced as a result of a certain condition (for example, a genetic mutation), physiological or environmental factors, which leads to a decrease or deficiency of LKL produced in the body. The LAL obtained according to the invention can also be used to treat other conditions such as atherosclerosis, liver obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatosis (NASH) and cirrhosis. The LAL obtained according to the invention can also be used to treat other conditions, such as those described in US Pat. 6849257 of February 1, 2005 (the contents of which are incorporated herein in their entirety by reference), US Patent Publication No. 2009/0297496 dated December 3, 2009; US Patent Publication No. 2004/0223960 dated November 11, 2004; US Patent Publication No. 2007/0264249 of November 15, 2009, the contents of which (i.e. the contents of all four patent publications) are hereby incorporated by reference in their entirety.

It is also contemplated that the LAL obtained by the invention can be used to treat certain specific conditions, including pancreatitis, for example, chronic pancreatitis and / or acute pancreatitis, as well as alcohol-induced pancreatitis, such as alcohol-induced pancreatitis.

LCL obtained by any suitable method, such as the method described herein, is provided for the treatment of diseases caused by alcohol-induced cell damage, including, but not limited to, alcohol-induced cell damage that leads to the accumulation of lipid esters in body tissues, such as , but not limited to, the liver, spleen, intestines, and tissue of the cardiovascular system. The invention also provides for the treatment of malabsorption by administering LCL.

One aspect of the invention describes methods for treating patients, comprising administering to the patient a therapeutically effective amount of a composition comprising recombinant human LAL, as described herein. The patient may suffer or be diagnosed with any number of conditions, including conditions associated with LAL deficiency. In one embodiment, the therapeutically effective amount is represented by an amount that increases the number of red blood cells in the patient by the desired amount. It is envisaged that the LAL obtained according to the invention can be used to treat chronic kidney disease, for example, if the tissues do not produce lysosomal acid lipase.

It is also contemplated that LCL obtained by any suitable method can be used to treat patients with Tangier disease and hereditary hypoalphalipoproteinemia. Tangier's disease / hereditary hypoalphalipoproteinemia is associated with the accumulation of cholesterol esters in macrophages, which is accompanied by hepatosplenomegaly and / or lymphadenopathy along with low levels of high density lipoproteins (HDL), which can be treated by the administration of LCL. For example, not wanting to limit the invention to any particular theory or mechanism of action, it is believed that impaired LAL activity reduces ABCA1 expression, and the increased LAL activity obtained by administering LAL to a patient with Tangier disease / hereditary hypoalphalipoproteinemia will increase ABCA1 expression to overcome the effects of the ABCA1 gene with reduced functional activity as a result of polymorphism.

For the treatment of the condition, as a rule, the dose administered can vary greatly depending on known factors, such as age, state of health and weight of the recipient, type of competitive treatment, frequency of treatment, etc. Typically, the dose of active active substance may be from about 0.0001 to about 10 mg per kg of body weight. The exact dose, frequency of administration and time period can be determined by a person skilled in the art for the corresponding therapeutic protein.

In addition, it has been found that doses of 1 mg / kg or less may be effective in treating LAL deficiency. The present invention describes methods for treating conditions, including administering to a mammal (eg, a patient, mainly a human patient) a therapeutically effective dose of lysosomal acid lipase from once every 5 days to once every 25 days, for example, from once every 7 days to one times every 14 days. In one embodiment, the dose of lysosomal acid lipase is administered from about 0.1 mg to about 50 mg per kg body weight, for example, the dose may be from about 1 mg to about 5 mg per kg.

In a separate embodiment of the invention, methods are described for treating a condition by administering a dose of lysosomal acid lipase from about 0.1 mg to 1.0 mg per kg body weight in accordance with any mode of administration of a therapeutically effective dose, such as that described in the text of this application.

The invention describes methods for treating any complication of LAL deficiency that can be treated with a therapeutically effective dose of LAL. In one embodiment of the invention, malabsorption and growth disorders can be treated according to the methods described herein. In another embodiment of the invention, using the methods described herein, complications noted in patients with LAL deficiency in a patient can be treated, including but not limited to hepatomegaly and liver dysfunction.

The invention describes a treatment using recombinant LCL (e.g., recombinant human LCL), which can be obtained using any suitable protein expression system, transgenic mammals and birds, as is customary in science. Other protein expression systems may include, but are not limited to, cell culture, bacterial and plant systems.

The invention encompasses the administration of recombinant LCL as part of a pharmaceutically acceptable composition in any way that will achieve the desired therapeutic effect, as determined by a person skilled in the art. In one embodiment, LCL can be administered by intravenous infusion over a period of about five hours. For example, infusion can be facilitated by using an outpatient infusion pump attached to a vascular access port (e.g., Port-a-Cath).

The invention also includes monitoring of clinical and pathological signs of conditions, for example, Wolman’s disease and BNEC, in mammals (for example, a human patient). In one embodiment, the evaluation consists of, but is not limited to, the following: lipid analysis, chest x-ray, functional liver tests, stool analysis, plasma mevalonic acid level, immunogenicity, plasma lysosomal acid lipase level, chitotriosidase level, PARC, portal hypertension, anthropometry, volume and characteristics of the liver, spleen and gastrointestinal tract using, for example, a visualization method. For example, the above imaging method may consist of ultrasound, magnetic resonance imaging, and nuclear magnetic resonance spectroscopy.

EXAMPLES

The invention is further described by the following examples. The examples are presented for illustrative purposes only and are not intended or should not be construed as limiting the invention in any way.

Example 1

Construction of a vector (pALVIN-OVRl-I-hLAL-dSA) carrying the coding sequence of recombinant human lysosomal acid lipase (rhLAL)

The nucleotide sequence of the hLAL gene in the pALVIN-OVRl-I-hLAL-dSA vector encodes a protein that is identical to the amino acid sequence of the protein obtained using the human lysosomal acid lipase gene (GenBank Accession, NP_000226) (FIG. 1). Transcription of this sequence and subsequent translation of the resulting mRNA leads to the formation of a 399 amino acid precursor protein, which is processed into a mature protein of 378 amino acids, identical to human LAL (GenBank Accession, NP_000226) (FIG. 1) with the sequence SEQ ID NO: l. The expression of the hLAL gene (see FIG. 2 for the cDNA sequence) in this Example is controlled by non-coding elements derived from the egg albumin gene, including enhancer, promoter, intron and 5 ′ and 3 ′ untranslated sequences. The egg albumin gene produces egg albumin, the main constituent protein in egg white. The activity of the chicken egg albumin promoter is extremely specific in the cells of the oviduct of chickens that produce egg protein; expression in other tissues is minimal.

To obtain a replication-defective retrovirus (RDR) that stably integrates the hLAL transgene into the founder genome (XLL109), the plasmid vector pALVIN-OVRl-I-hLAL-dSA (FIG. 3A; the nucleotide sequence is shown in FIG. 4) was used. Such a plasmid vector includes retroviral nucleotide sequences necessary for virus RNA packaging, reverse transcription and integration, but does not contain intact sequences for the gag, pol and env genes of the virus. Methods for producing a retroviral vector and its use in subsequent transgenic procedures are described in the text of this application.

The retroviral region of pALVIN-OVRl-I-hLAL-dSA is based on the ALV pNLB vector. pNLB has been modified so that LTRs are self-inactivating (SIN) (FIG. 3B). To achieve this, 273 bp from 3 'LTRs were removed, which include the enhancer and the CAAT box of the U3 section. Whereas the inactivated U3 region at the 3 ′ end of the retroviral sequence serves as a template for the new U3 region present at the 5 ′ end of the integrated provirus, 5 ′ LTR is also usually inactivated. The deletion of LTR sequences in the SIN vector reduces the interference of the promoter relative to the internal LTR promoter and minimizes the possibility of sequence recombination to form a replication-competent retrovirus. The new vector is called pALVIN for the ALV inactivation vector.

Down from the 5 'LTR is a portion of the gag and env coding sequences that were brought from the pNLB vector. In pALVIN-OVRl-I-hLAL-dSA, a small portion (12%) of the gag precursor protein sequence (55% of the mature p19 peptide sequence) and a small portion (1.7%) of the RAV2 precursor sequence (GenBank Accession, AF033808) remain. Such branched regions of gag and env are unable to produce the functional proteins necessary to create a replication-competent retrovirus (Cosset, 1991).

Chicken egg albumin gene transcription and translation controls were inserted into pALVIN to create pALVIN-OV-ll-I (the sequence is shown in FIG. 6; SEQ ID NO: 8). The first pALVIN-OV-1.1-1 region consists of the adjacent region of the chicken egg albumin gene, which includes the 1.1 kb proximal promoter region, the first exon, the first intron, and part of the ^second exon. The next section is represented by a spacer insertion fragment, which takes the place of the coding sequences of egg albumin protein. The spacer is followed by a 3′-untranslated region (UTR) of the chicken egg albumin gene, which includes sequences that facilitate proper mRNA processing, including polyadenylation. In general, the spacer fragment is replaced by DNA fragments encoding the desired protein in the case of hLAL. The result is a vector with bispecific elements that facilitate the regulated transcriptional expression and translation of mRNA in the oviduct of transgenic chickens, which closely mimics the regulation of endogenous egg albumin mRNA and ensures high expression of the protein of interest in egg protein.

Vector pALVIN-OV-l.l-I includes the first intron of the egg albumin gene. Considering that the intron is prone to splicing during generation and packaging of the retroviral RNA genome, we inserted the expression cassette in the opposite position relative to LTR. In this case, the intron is not recognized by retroviral RNA and is packaged without splicing. For convenience, all cards in this document are presented in such a way that the LTR is in the opposite position and the expression cassette is in front or clockwise.

pALVIN-OV-l.l-I is the base vector into which the hLAL coding sequence (CDS) has been inserted. Integrated DNA Technologies, Coralville, Iowa, (see FIGS. 7 and 8; SEQ ID NO: 9 and 10) synthesized two DNA fragments — the hLAL adapter and Syn hLAL — which make up the hLAL CDS and the sequences required for compatibility with pALVIN-OV-ll-I. The 229 bp Hpal / BamHI fragment of the hLAL adapter and the 1113 bp BamHI / BstBI Syn hLAL fragment were inserted into the 7882 Hpal / BstBI pALVIN-OV-ll-I fragment, thereby replacing the spacer region in the hLAL CDS and creating pALVIN-OV-ll-l -hLAL.

It has been revealed that there is a hidden splicing site in the hLAL CDS antisense strand that prevents packaging of intact retroviral RNA. The hidden splicing site was removed by changing the DNA sequence without changing the amino acid sequence of hLAL. This change was made using polymerase chain reaction amplification of sections 232 to 534 pALVIN-OV-ll-I-hLAL with primer 5'-AGAAACTGAGAGTGTCTTAT-3 '(SEQ ID NO: 12) and primer 5'-TGACAGCTGTGGATCCAGAAACAAAC SEQ ID NO: 13) by creating an amplicon of 329 bp. Such an amplicon was cleaved with BamHI and SexAI and ligated with 8940 bp BamHI / SexAI pALVIN-OV-l.l-I-hLAL to create pALVIN-OV-1.1 -I-hLAL-dSA.

A putative promoter / enhancer that contains the chicken egg albumin gene DNAse III (DHSIII) hypersensitivity site (-3819 to -2169 relative to the start site of the OV promoter) (Kaye, Bellard et al. 1984) was inserted into pALVIN-OV-1.1-I -hLAL-dSA to create pALVIN-OVRl-I-hLAL-dSA. This was done as follows: a DNA fragment comprising a DHSIII enhancer and a 1.1 kb OV proximal promoter, called the OVR1 promoter (see FIG. 9; and SEQ ID NO: 11) was isolated by Xhol and BlpI cleavage. To facilitate subcloning, a PCR adapter fragment pSIN-OV-1.1-1 was obtained by PCR amplification of the region from 6752 to 7974 pALVIN-OV-ll-I with primers 5'-GCCGCTCGAGCGAGGAATAATAAAAAAATT-3 '(SEQ ID NO: 14) and 5' -TCCGCGCACATTTCCCCGAA-3 '(SEQ ID NO: 15) followed by cleavage of NgoMI and Xhol. A 2772 bp fragment of the XhoI / BlpI promoter OVR1 and a 1067 bp NgoMI / XhoI PCR fragment pSIN-OV-1.1-I were inserted into a 7043 bp NgoMI / BlpI pALVIN-OV-ll-I-hLAL-dSA, thereby creating pALVIN-0VR1 -I-hLAL-dSA (see FIG. 10, which shows the scheme for obtaining pALVIN-0VRl-I-hLAL-dSA). The design of the retroviral vector segment designated pALVIN (aka p AVIJCR-A395.22.3.1-KM or pALV-SIN) is described in US Patent Application 2008/0064862.

In addition, the preparation of LAL in accordance with the invention and the use of the promoter and / or vector described in US Pat. 2008/0064862 dated March 13, 2008, the contents of which are hereby incorporated by reference in their entirety.

Example 2

Getting a viral particle

Using the following retroviral transgenesis method, a transgenic male founder of the G0 XLL109 line was created, carrying the hLAL transgene in its genome. Replication-defective viral particles carrying the pALVIN-OVRl-I-hLAL-dSA vector were obtained by transiently transfecting an immortalized chicken fibroblast cell line. Such chicken fibroblast cells were simultaneously transfected with three plasmids pALVIN-OVRl-I-hLAL-dSA, pCMV-gag-pol and pCMV-VSV-G. pCMV-gag-pol expressing the gag and pol genes of avian leukemia virus RAVI strain. pCMV-VSV-G expresses the vesicular stomatitis virus envelope protein. 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. The medium was collected 48 hours after transfection, filtered through a 0.45 μm filter (Millipore) and concentrated by ultracentrifugation. A concentrated retrovirus carrying the ALVIN-OVRl-I-hLAL-dSA transgene was collected and used in early transduction of embryos. It should be noted that since “p” denotes the shape of the plasmid of the vector, “p” is not present in the transgene designations as soon as the transgene becomes a packaged vector or integrated transgene.

Example 3

Embryo Transgenesis

The integration of the ALVIN-OVRl-I-hLAL-dSA expression cassette into the embryo genome was achieved by early transduction of the embryos (Speksnijder and Ivarie, 2000). Freshly laid fertilized white Leghorn eggs were obtained from a bred colony of birds. A hole was made in the shell to provide access to the embryo. Seven microliters of concentrated replication-deficient retrovirus particles carrying the ALVIN-OVRl-I-hLAL-dSA expression cassette described above were introduced into the subgerminal cavity of the embryo. The eggs were sealed with hot glue and incubated with hatching under standard conditions. Prognosis resulting from these injections was marked with individual markers in the litter for identification and traceability. Blood samples of the offspring were transgene positive in real-time PCR analysis for the hLAL transgene using PCR primers specific for the hLAL coding sequence (as described below). This allowed us to identify the success of the transgenesis procedure. Real-time PCR analysis for the hLAL transgene uses the Taqman® chemical kit (Applied Biosystems). Forward and reverse primers were presented 5'-ACGACTGGCTTGCAGATGTCT-3 '(SEQ ID NO: 16) and 5'-ConverterAAATGAAGTCAAGATGCT-3' (SEQ ID NO: 17), respectively. The Taqman® probe sequence was represented by 5'-CCGGAATGCTCTCATGGAACACCAA-3 '(SEQ ID NO: 18) and was labeled FAM (as a radiator) at the 5'-end and Iowa Black (as a quencher) at the 3'-end. Primers, a probe, and 1 μl of extracted DNA were added to 30 μl of Taqman® Universal Master Blend (Applied Biosystems). Control reactions included various dilutions of a plasmid carrying the hLAL sequence and wild-type chicken DNA (data not shown). Applied Biosystems 7500 Fast used standard cyclic parameters for the real-time PCR system.

Example 4

Identification of the founder of G0

Semen was taken from mature males, and DNA was extracted and analyzed using real-time PCR for hLAL. The number of transgenic copies in each sample was estimated using known standards (plasmids carrying the hLAL gene) mixed with negative control sperm DNA. The DNA content of the transgenic cassette in the male XLL109 was at a level that ensures transgene transmission in this offspring, which was evaluated by real-time PCR. Such a male XLL109 was designated as the transgenic founder of G0 and was bred with non-transgenic chickens to produce hemizygous transgenic G1 chickens.

Example 5

Reproduction and characterization of hemizygous birds G1

Progeny from transgenic founder XLL109 was tested for the presence of the transgene in the blood cell DNA using real-time PCR analysis on hLAL. Blood was collected from 1-2 week offspring, and DNA was extracted using a high throughput method (Harvey et al., 2002). To facilitate high throughput screening, DNA was not quantified prior to Taqman analysis. Typically, 1 μl of DNA contains from 50 to 400 ng of DNA, which is sufficient to obtain a positive amplification signal. A total of 1322 chickens fertilized by XLL109 were tested, and blood was collected from positive offspring and retested for confirmation. According to PCR results, 22 offspring were positive for the ALVIN-OVR1-I-hLAL-dSA transgene. An example of Taqman results is presented in FIG. eleven.

Example 6

High expression line identification and characterization

One of the G1 1LL7466 hens laid eggs with significantly elevated levels of rhLAL protein in egg protein compared to other G1 chickens. Southern blotting and fertilization by G1 males was performed for 1LL7466 to identify which males have the same integration site as chickens with high expression. The digestion was carried out with rextractase, which cleaved only once in the transgene (BlpI), and Southern blotting was performed with a probe having a segment of the egg albumin promoter or hLAL coding sequence (Fig. 12A-D). The position of the 2nd restriction site located in the flanking genomic region varies depending on the integration site. Thus, the BlpI band size determined by the OV probe or hLAL probe is unique for each received line.

The OV probe detected a separate 4.1 kb band in the DNA digested by Blpl and obtained from wild-type chickens, which corresponded to the expected size of the BlpI segment in the endogenous egg albumin gene of the chicken genome (FIGS. 12B and 12D). The second band of 4.3 kb was determined in chicken 1LL7466, which corresponded to the transgenic band. Three additional female siblings ILL 10409, 1LL10686 and 1LL12058 and three additional male siblings 1LL8922, 1LL9330 and ILL11217 had a 4.3 kb band, which indicated that such siblings could belong to the same line (FIGS. 12B and 12D).

As expected, the hLAL probe did not reveal DNA bands in wild-type chickens, as the DNA sequence of the lysosomal acid lipase gene in chickens and the coding sequence of the recombinant human lysosomal acid lipase are essentially differentiated, which does not allow hybridization to occur under the conditions used for this Southern blotting (FIG. 12C). The hLAL probe detected a single ~ 10.6 kb band in the genomic DNA digested by Blpl and obtained from the same chickens that were positive for the 4.3 kb band detected by the OV probe, which indicates that these 7 G1 chickens have the same integration site and therefore belong to the same line.

No other bands were determined, which indicates that 1LL7466, ILL10409, 1LL10686, 1LL12058, 1LL8922, 1LL9330 and 1LL11217 had the same integration site.

Southern blotting also showed that the transgene was integrated because the bands determined by the OV and hLAL probes had different sizes and their size exceeded the size obtained only in the presence of a transgene. In FIG. 12A, a map is shown showing the predicted structure of an integrated transgene and BlpI site positions in flanked genomic regions.

To confirm the intactness of the transgene, two steps were performed. First, the hLAL coding sequence was isolated by PCR from 1LL7466. PCR products were sequenced in both bands from the hLAL start codon to the stop codon. The DNA sequence was the same as expected, indicating no change in the DNA sequence of the coding regions of the transgene. Secondly, Southern blotting was performed using the restriction enzyme ApaLI, which cleaves the intact transgene into 2 segments, 3.6 and 3.8 kb (FIG. 13A). Bands 3.6 and 3.8 kb were determined using genomic DNA cleaved with ApaLI and obtained from Gl, which indicated that the transgene was integrated in a completely intact form (FIG. 13B).

Example 7

Reproduction and characterization G2

In FIG. 14 shows the hLAL line G2 received from one founder G0 XLL109. At the G1 stage, the transgene was characterized with respect to copy number, integrity, hLAL sequence and integration site, as a result of which seven G1 transgenes (four chicken and three roosters) were identified and characterized.

Reproduction of G2 was accompanied by artificial insemination of non-transgenic chickens with sperm taken from males G1 1LL8922, 1LL9330 and 1LL11217 (FIG. 14). Each fertilized chicken laid eggs, and the resulting offspring were kept separate from other offspring. Hatching offspring were tested for the presence of the hLAL transgene using real-time PCR for hLAL. Given that the founders of G1 were hemizygotes for the transgene, half of the offspring was expected to be transgenic G2. Of the 610 G2 descendants analyzed at a given point in time, 330 or 54% were transgenic.

Example 8

Genetic analysis of hLAL birds

After each chicken G2 was identified using real-time hLAL PCR and blood DNA analysis, the gene line was subjected to the following genetic analyzes: the hLAL gene was PCR amplified using blood DNA and sequenced to confirm 100% homology to the human sequence; the transgene integration site has been confirmed by integrating the PCR site as described above. Sequencing of PCR and analysis of the integration site was performed on each chicken in the line of reproducers <10; 10% of chickens (minimum 10) for a line of reproducers 11-100 hens; 5% of chickens (minimum 10) for a line of reproducers 101-1000 chickens; 1% of chickens (minimum 50) for the line of reproducers 1001-10000 chickens; 0.1% of chickens (minimum 100) for a breeding line> 10001 chickens. Detailed records were taken at each stage of the growth and reproduction phases.

Example 9

Egg hLAL purification

Egg white (YB) containing LAL was solubilized at pH 6 overnight and purified by centrifugation (or deep filtration) with a 0.2 μm filter. YaB was adjusted with a buffer of 1 M NaOAc (pH 4) to pH 6.

Purified YB was loaded onto a Phenyl-HIC column (YB: column size = 2: 1), in which equilibrium was established with 20 mM phosphate / 137 mM NaCl buffer (pH 6). After loading was completed, the column was washed with equilibration buffer and 5 mM phosphate buffer (pH 6). LAL was eluted with 30% propylene glycol with 5 mM Tris buffer (pH 7.2).

The eluted LKL fraction was adjusted to pH 5 using 1 M acid and then loaded onto a GigaCap S column (YB: column size = 10: l). Column equilibrium was established using 50 mM NaOAc buffer (pH 5). After loading was completed, the column was washed with buffer to establish equilibrium. LAL was eluted using 50 mM NaOAc / 60 mM NaCl (pH 5).

The LCL fraction from the GigaCap S column was adjusted to pH 6 using 1 M Tris buffer and then loaded onto the Butyl-HIC column (YB: column size = 10: 1). The column was equilibrated using 20 mM phosphate / 137 mM NaCl buffer (pH 6). After loading was completed, the column was washed with equilibration buffer and 5 mM phosphate buffer (pH 6). Pure LAL was eluted with 50% propylene glycol with 5 mM Tris buffer (pH 7.2). FIG. 15 depicts the steps for purifying hLAL from egg white.

Example 10

Carbohydrate analysis of hLAL obtained from transgenic birds

Oligosaccharide structures were determined in human LAL obtained from birds, using the following analysis method, which should be well known to specialists in this field.

200 mg was digested with trypsin and chemotrypsin for 18 hours at 37 ° C in 0.1 M Tris-HCl, pH 8.2, containing 1 mM CaCl ₂ . The cleavage products were enriched and freed from contamination using a Sep-Pak C18 cartridge column. After enrichment, the glycopeptides were digested with 2 MKL of PNHase F (7.5 u / ml) in 50 μl of 20 mM sodium phosphate buffer, pH 7.5, for 18 hours at 37 ° C. The released oligosaccharides were separated from the peptide and enzyme by passing them through a Sep-Pak C18 cartridge column.

The glycan fraction was dissolved in dimethyl sulfoxide and then permethylated based on the Anumula and Taylor method (Anumula and Taylor, 1992). The reaction was quenched by adding water, and per-O-methylated carbohydrates were extracted with dichloromethane. Per-O-methylated glycans were dried under a stream of nitrogen.

The MALDI / TOF-MS analysis (time-of-flight mass spectrometry with laser ionization and liquid matrix desorption) was performed in a reflector-positive ionization mode using alpha-dihydroxybenzoic acid (DHBA, 20 mg / ml solution in 50% methanol: water) in as a matrix. All spectra were obtained using Microflex LRF (Bruker).

The MALDI-TOF-MS and ESI MS / MS (tandem electrospray ionization mass spectrometry) analyzes were performed on oligosaccharides after release from the peptide backbone and purification, as described in science. Samples of certain types of oligosaccharides were also digested with specific enzymes, and the degradation products were analyzed by HPLC as described in science.

It is estimated that there are approximately six N-linked glycosylation sites in human LAL. See Zschenker, et al (2005) J. Biochem., Vol 137, p 387-394, the contents of which are incorporated herein in their entireties by reference. This reference also indicates that an O-linked glycosylation site may exist in human LAL. The identified N-linked oligosaccharide structures are shown in FIG. 16.

The data showed that most or all of these structures were identified as N-linked glycosylated structures in the LCL obtained according to the invention (FIG. 16). For example, as it turned out, A-n is attached to the LCL obtained according to the invention. For example, O-n is attached to the LCL obtained according to the invention. For example, at least one Bn, Cn and Dn are attached to the LCL obtained according to the invention. For example, at least one E-n and F-n are attached to the LCL obtained according to the invention. For example, at least one In and Jn are attached to the LCL obtained according to the invention. For example, at least one K-n and L-n are attached to the LCL obtained according to the invention. For example, at least one Mn and Nn are attached to the LCL obtained according to the invention. For example, as it turned out, Gn is attached to the LCL obtained according to the invention. For example, as it turned out, H-n is attached to the LCL obtained according to the invention.

Example 11

N-glycan LCL species obtained from transgenic birds

Purified LCL samples obtained from transgenic birds (600 μg / sample) were dialyzed using a Tube-O-Dialyzer dialyzer (4.0 kDa cut-off membrane; G Biosciences) with respect to nanopure water at 4 ° C for 24 hours to remove salts and other impurities. Nanopure water was replaced four times during the entire dialysis period.

After dialysis, each sample was divided into three aliquots: ~ 1/4 of the weight of the sample for analysis for neutral and amino sugars, ~ 1/4 of the weight of the sample for analysis for mannose-6-phosphate, and ~ 1/2 of the weight of the sample for analysis on the oligosaccharide profile. An aliquot for neutral and amino sugar analysis was hydrolyzed with 2 N trifluoroacetic acid (TFA) at 100 ° C for 4 hours, and an aliquot for analysis of mannose-6-phosphate was hydrolyzed with 6.75 N TFA at 100 ° C for 1 , 5 hours. Then the hydrolysates were dried thanks to N ₂ , re-dissolved in 50 μl of H ₂ O, subjected to ultrasound for 7 minutes on ice and transferred to an injection vial. Samples with neutral and amino sugars were diluted 2 times, since the peaks obtained from the initially diluted hydrolysates were too large.

A mixture of standards for neutral and amino sugars, as well as mannose-6-phosphate with a known number of moles, was hydrolyzed in the same way for the same time as the samples. To establish equilibrium during calibration, four concentrations of a standard mixture of neutral and amino sugars were prepared (Fuc and GalNAc, 0.2, 0.4, 0.8, and 1.6 nmol per 10 μl; GlcNAc, 0.5, 1.0, 2 , 0 and 4.0 nmol per 10 pl; Gal and Man, 0.3, 0.6, 1.2 and 2.4 nmol per 10 pl; and Glc, 0.1, 0.2, 0.4 and 0.8 nmol per 10 pl) and mannose-6-phosphate (640, 1280, 2560, 5120 picomol per 10 pl). The number of moles of each sugar in the sample was quantified by linear interpolation from the calibration equation.

Neutral and amino sugars, as well as mannose-6-phosphate, were analyzed by VEAOH using a Dionex ICS3000 system equipped with a gradient pump, an electrochemical detector, and an automatic sampler. Separate neutral and amino sugars, as well as mannose-6-phosphate, were separated by a Dionex CarboPac PA20 analytical column (3 × 150 mm) with an amine trap. Gradient programs used solvents A, degassed nanopure water, and B, 200 mM NaOH for neutral and amino sugars, and C, 100 mM NaOH and D, 1 M sodium acetate in 100 mM NaOH for mannose-6-phosphate. Administration (10 μl / injection) was performed every 40 minutes to determine neutral and amino sugar, and every 35 minutes to determine mannose-6-phosphate. All methods were based on protocols described by Hardy and Townsend (Hardy, M. R., and Townsend, R. R., "High-pH anion-exchange chromatography of glycoprotein-derived carbohydrates", 1994, Methods Enzymol. 230: 208-225). Instrument control and data acquisition was performed using Dionex chromeleon software. The results are shown in Table 1 below. The control sample is represented by an ovomucoid purified from ulcer.

Structural Features of LKL

LCL has 6 potential sites in its amino acid sequence for N-linked glycosylation, namely Asn ³⁶ , Asn ⁷² , Asn ^101, Asn ¹⁶¹ , Asn ^273, and Asn ³²¹ . Five of them, Asn ³⁶ , Asn ¹⁰¹ , Asn ¹⁶¹ , Asn ²⁷³ and Asn ^321, were glycosylated, while Asn ⁷² remained aglycosylated or essentially aglycosylated (essentially aglycosylated means a mixture of LCL molecules in which several Asn ^{72 are} glycosylated compared to Asn ³⁶ , Asn ¹⁰¹ , Asn ¹⁶¹ , Asn ²⁷³ and Asn ³²¹ ). Accordingly, one aspect of the invention provides LAL (eg, human LAL) that is aglycosylated and / or substantially aglycosylated in Asn ⁷² , as well as the preparation and use of such LAL. However, LAL with glycosylated Asn ^{72 is} also included in the scope of this invention. N-glycosylated structures mainly consist of a mixture of bi-, tri- and tetraantennary structures with N-acetylglucosamine, mannose and mannose-6-phosphate (M6P) as the main sugars. As it turned out, each site has a characteristic set of structures (Table 2 and FIG. 17), which is one aspect of the invention. For example, modified M6P N-glycans are located in at least Asn ¹⁰¹ , Asn ¹⁶¹ and Asn ²⁷³ . The non-phosphorylated structures are represented by typical N-glycans located on endogenous egg white proteins. As determined by the deficiency of N-acetylgalactosamine (GalNac), no O-linked glycans were detected. Sialic acid was also not detected, which corresponds to the previously determined N-glycan structures of other endogenous and exogenous proteins obtained in accordance with the invention. The invention includes LALs glycosylated by one or more of the oligosaccharide structures described herein.

Ways

The composition of monosaccharides, including neutral, amino and M6P sugars, was quantified and qualitatively determined by amperometry using high pH anion exchange chromatography (HPAEC-PAD).

The structures of the predominant glycans were determined using data from various mass spectrometry methods (MALDI-TOF, NSI-MS / MS and LC-MS glycopeptides).

MALDI-TOF was used to determine neutral N-glycans, and such a method was able to detect phosphorylated N-glycans (FIG. 18). The NSI-MS / MS method was used to determine the nature of the smaller peaks of the MALDI-TOF spectrum, some of which were characteristic of phosphorylated N-glycans (FIG. 19). Attempts to improve the ability of MALDI-TOF to detect phosphorylated N-glycans have been unsuccessful.

LC / MS glycopeptides were able to determine neutral and phosphorylated structures, as well as the position of specific structures in the amino acid sequence of LAL (data are summarized in FIG. 17 and Table 2).

To determine which peaks in the HPAEC-PAD chromatogram are caused by phosphorylated N-glycans, LAL was phosphatase treated and analyzed (Figure 3). Peaks in groups C and D reduced the area under the curve (PPC), while the peak from group A was more prominent. Peaks from group B did not change the proportion relative to other peaks. Based on the knowledge that the retention time is proportional to the degree of charge (due to phosphorylation or sialylation), it was found that group C consists of N-glycans with one phosphate (mono M6P) and group D consists of N-glycans with two phosphates (bis- M6P).

The retention time was also influenced by the composition and the relative structural position of the neutral and amino saccharides. Such examples include the presence of galactose, the presence of bisecting GlcNac, and the degree of substitution of GlcNac. These factors contribute to the formation of multiple peaks in the HPAEC-PAD chromatogram.

Example 12

Analysis of the activity of an in vitro enzyme obtained as hLAL in transgenic birds as part of egg white

The activity of lysosomal acid lipase in egg protein was determined using analysis of the fluorogenic substrate of 4-methylumbelliferryl oleate, as essentially described in Yan et al. (2006), American Journal of Pathology, Vol. 169, No. 3, p 916-926, the contents of which are incorporated herein in their entireties by reference.

A stock solution of 4-methylumbelliferryl oleate (4-MUO) was prepared, consisting of 2.5 mM 4-MUO in 4% Triton X-100. The analysis was carried out carefully in a microtiter plate, in each well of which 62.5 μl 0.2 M sodium citrate (pH 5.5) was added to 0.01% Tween 80, 12.5 cells of an egg white sample and 25 μl 2.5 mM 4-MUO. Changes in fluorescence were monitored for 30 minutes at 37 ° C using a Bio-Tek Synergy HT fluometric plate reader (excitation at 360 nm and emission at 460 nm). Before analysis, the egg protein containing hLKL was diluted to the concentration of the enzyme, which led to a linear reaction lasting at least 30 minutes. The reaction was stopped with 50 μl of 0.75 M Tris-HCl, pH 8.0, and an end-point fluorescence signal was measured using the above plate reader (excitation at 360 nm and emission at 460 nm).

Activity units were determined using 4-methylumbelliferryl as a standard. One unit (U) is defined as the amount of enzyme that leads to the formation of 1 μmol of 4-umbelliferril / min under the above analysis conditions. Egg negative protein containing no hLLC was used as a negative control.

Samples of egg protein, which were positive for hLKL, contained from 1 Unit. up to 100 units activity per ml of egg white. Egg protein from 21 chicken G1 was analyzed. Egg protein from 10 studied chickens had positive hLCL activity.

Example 13

In vitro analysis of LCL obtained from transgenic birds

The ability of LAL produced in the oviduct cells of transgenic birds (indicated as “SBC-102”, “obtained from birds LAL”, “LAL” or “hLCL”) to bind to cells and internalize in the lysosomal compartment was studied in vitro using macrophages and fibroblasts. Upon incubation in macrophage cells, the localization of SBC-102 with a fluorescent label in lysosomes was detected. This effect could be achieved using a competitor of the mannose polysaccharide involving the N-acetylglucosamine / mannose receptor (GlcNAc / mannose) as a mechanism for recognition and uptake by such cells. SBC-102 increased cell-associated LAL activity in human LAL-deficient fibroblasts and normal mouse fibroblasts after in vitro incubation, indicating that exposure to SBC-102 could significantly replace the missing enzymatic activity.

Mannose-6-phosphate (M6P) is present in the SBC-102 oligosaccharide structures, which have been shown to be involved in the delivery of lysosomal enzymes to a variety of cell types through the ubiquitous M6P receptor.

LAL was purified from transgenic chicken egg white. Oregon Green NHS was obtained from Invitrogen ™ (No. 0-10241). From ATCC, the macrophage line of rat alveoli NR8383 and the mouse fibroblast line NIH-3T3 were obtained. LKL deficient fibroblasts with Wolman disease were obtained from the Coriell Institute for Medical Research, and Invitrogen ™ was presented to LysoTracker® Red.

Labeling of the enzyme: 4 mg of LCL obtained from transgenic birds in the FSB was labeled Oregon Green according to the manufacturer's recommendations, and the reaction was subsequently dialyzed relative to the FSB followed by concentration.

Macrophage capture: Fluorescence-labeled LCL obtained from transgenic birds (5 μg / ml) and LysoTracker® Red were incubated with NR8383 cells for 2 hours. Cells were examined by cofocal fluorescence microscopy using a sequential scanning mode at 488 nm and then at 514 nm.

Competitive inhibition with mannan: SBC-102 with a fluorescent label (5 μg / ml) and mannan were incubated with NR8383 cells for 2 hours. Cells were trypsinized, and LAL uptake was measured by activated fluorescence cell sorting using the median fluorescence intensity as the endpoint.

The ability of LAL obtained from a transgenic bird to be captured and subsequently incorporated into the lysosomes of target cells was investigated using the macrophage cell line NR8383. Fluorescently labeled LCL obtained from transgenic birds and the LysoTracker® Red lysosomal marker (Invitrogen ™) were incubated with cells for 2 hours. Subsequently, the solocalization of LCL obtained from transgenic birds and the lysosomal marker in lysosomes was studied by confocal fluorescence microscopy and sequential scanning mode (FIG. 20). LAL was localized in lysosomes, which is consistent with similar in vitro studies using rhLCL from various sources.

The specificity of the binding of LAL obtained from transgenic birds to the GlcNAc / mannose receptor was evaluated by competitive binding assays using the macrophage cell line NR8383 (FIG. 21). LCL obtained from transgenic birds with a fluorescent label (Oregon Green) in an amount of 5 μg / ml and various concentrations of mannose-containing oligosaccharide, mannan, were co-incubated with cells for 2 hours. The relative inhibition of LAL uptake obtained in transgenic birds by mannan compared to the control without mannan was quantified by analyzing the sorting of fluorescence-activated cells using the median of fluorescence intensity as the endpoint. Dose-dependent mannose inhibition of LAL binding / uptake obtained from transgenic birds was noted, which corresponded to the LKL interaction obtained from transgenic birds: GlcNAcR.

In addition, mannose-6-phosphate-mediated uptake of fibroblasts in cells was proven by experiments on competitive binding to mannose-6-phosphate (data not shown).

The ability of LAL, obtained from transgenic birds, to increase LAL activity in cells was studied using cells with a normal content and deficiency of LAL in vitro. Fibroblasts isolated from a patient with Wolman disease and normal mouse fibroblasts (NIH-3T3) were incubated in the presence of LAL obtained from transgenic birds at concentrations of 0, 0.16 or 0.5 μg / ml for 5 hours. Cells were then washed to remove the non-specific signal, and cell lysates were analyzed for LAL activity using 4-MUO substrate. Endogenous cell-associated activity was lower in Wolman's fibroblasts compared to NIH-3T3, and in cells of both types after incubation with LAL obtained in transgenic birds, a dose-dependent increase in activity was observed (FIG. 22).

Example 14

In vivo analysis of LCL obtained from transgenic birds

Losh deficient Yoshida rats (i.e., homozygous) (see Kuriyama et al. (1990), Journal of Lipid Research, vol. 31, p 1605-1611; Nakagawa et al., (1995) Journal of Lipid Research , vol. 36, p 2212-2218; and Yoshida and Kuriyama (1990) Laboratory Animal Science, vol. 40, p 486-489) were exposed to SBC-102 (5 mg / kg, w / w) or placebo once per week for 4 weeks, starting at the age of 4 weeks. For each administration, SBC-102 was injected into the tail vein of rats in two equal doses (2.5 mg / kg) with an interval of 30 minutes. Rats and age-appropriate wild-type controls were examined one week after the final dose. Analyzes were performed in triplicate.

An extensive pathological examination of animals treated with SBC-102 revealed normalization of liver color, in addition to reducing organ size. Rats treated with SBC-102 had normal histological liver parameters with a stark contrast to a significant accumulation of foamy macrophages in animals that were administered vehicle (data not shown). Serum alanine and aspartate aminotransferase levels increased in rats with LAL ^{- / -} were also reduced in rats that were injected with SBC-102 (data not shown).

The mass of internal organs and tissues was determined for each rat, and the data are presented in FIG. 23. The size of the organs is presented as a percentage of body weight determined at the age of 8 weeks in rats LKL ^{- / -} and LKL ^{+ / +} after weekly administration of the vehicle or SBC-102 at a dose of 5 mg / kg for 4 weeks.

The body weight of Yoshida rats treated with SBC-102 or vehicle was compared with the weight of wild-type rats, as indicated in FIG. 24. SBC-102 (5 mg / kg) or vehicle was administered by intravenous injection in the form of a single dose or paired doses (administered over a period of 4 hours) to rats LKL ^{- / -} . Rats LKL ^{+ / +} served as a litter control of the appropriate age.

Example 15

Triglyceride Analysis

Triglycerides were analyzed on liver and spleen tissue taken from wild type animals, homozygous with placebo and homozygous with SBC-102. Triglyceride level analyzes were performed using standard methods (i.e., MBL International's Triglyceride Quantification Kit Catalog # JM-K622-100), and were performed in triplicate.

Substrate Levels in the Liver

FIG. 25 displays liver cholesterol, cholesterol esters, and triglycerides at 8 weeks of age in WT rats and LAL deficient rats after weekly administration of the vehicle or SBC-102 at a dose of 5 mg / kg ^-1 for 4 weeks.

Example 16

Dose Response Study

Based on the above studies, the pharmacodynamic (PD) effects of a number of doses and the dose schedule (once a week and once every two weeks) of LCL (“SBC-102”) were studied in LKL ^{- / -} rats. During such studies, SBC-102 was administered by iv injection at doses of 0.2, 1, 3 and 5 mg / kg once every 2 weeks or 0.35, 1 and 5 mg / kg once a week for 1 months starting from the age of 4 weeks. The results showed an improvement in body mass gain (BW) (FIG. 26), organomegaly (FIG. 27) and substrate levels in tissues (FIG. 28). With increasing doses of SBC-102, serum transaminase levels were also reduced, with levels reaching substantially wild-type levels using maximum doses.

Example 17

The introduction of recombinant LCL in a rat model

The effect of a repeated dose of recombinant human lysosomal acid lipase (LAL) on body weight, tissue triglycerides and cholesterol, hepatomegaly, splenomegaly, lymphadenopathy, intestinal weight and other parameters was evaluated for Donryu rats with LAL deficiency described by Yoshida and Kuriyama (1990) Laboratory Animal Science, vol. 40, p 486-489 (see also Kuriyama et al. (1990) Journal of Lipid Research, vol. 31, p 1605-1611; Nakagawa et al., (1995) Journal of Lipid Research, vol. 36, p 2212- 2218), the content is hereby incorporated by reference in its entirety.

At 4 weeks of age, Donryu rats homozygous for the absence of LAL (LAL ^{- / -} ) were divided into groups with doses of recombinant human LAL obtained from transgenic chickens via the oviduct system, or placebo. Litter of wild type rats of the appropriate age was used as a control. In rats, LKL ^{- / -} doses were administered once a week for four weeks (a total of 4 doses) or once every two weeks for four weeks (a total of 2 doses) by administration to the tail vein as a single dose or two equal doses with an interval of 30 minutes. Doses of LCL were 1 mg / kg or 5 mg / kg. The dose schedule is shown in Table 4. Diphenhydramine (5 mg / kg) was previously administered to rats to counteract the potential anaphylactic reaction, and this procedure is based on previous experiments in animal models during enzyme replacement therapy to treat lysosomal accumulation disease (Shull et al . (1994) Proceedings of the National Academy of Science, vol. 91, p. 12937; Bielicki et al. (1999) The Journal of Biological Chemistry, 274, p. 36335; Vogler et al. (1999) Pediatric Research, 45 , p.838), the contents are hereby incorporated by reference in their entireties.

FIG. Figure 29 shows the daily progress in weight gain in rats given 1 mg / kg LKL per week or 5 mg / kg LKL per week or 5 mg / kg LKL once every two weeks. The figure shows that there is a slight or no difference in the therapeutic effect between two different doses and the frequency of administration of doses.

Anatomopathological study of rats LKL ^{- / -} , which were introduced recombinant LKL

At the end of the study described in Example 1, the animals participating in the study were humanely euthanized for extensive anatomopathological analysis, histopathology, and clinical chemistry. Extensive autopsy included examination of the inner surface of the body, all openings, as well as the cranial, thoracic abdominal cavity and their contents. The mass of internal organs was determined for rats, and organs and tissues were collected and fixed in 10% neutral buffered formalin. After fixation, the tissues were processed and histological slides were prepared and studied with hematoxylin and eosin staining.

Extensive anatomical examination of the treated animals revealed essentially normalization of the size and color of the liver, as indicated in FIG. 30. The ratios between organs and body weight were determined, and a decrease in the relative organ size was shown for the liver, spleen, mesenteric tissue, duodenum, small intestine, and ileum in successfully treated animals that were dissected compared to the treated rats placebo (FIG. 23). A histopathological analysis of liver tissue in rats treated with LAL showed essentially normal histological parameters in stark contrast to a significant accumulation of foamy macrophages in animals treated with placebo (FIG. 30).

Example 18

Treatment of Wolman disease (BV) by the introduction of recombinant LCL

At the age of 7 weeks, a female patient was admitted to the hospital due to difficulty in weight gain and poor progress from birth. At the initial physical examination, the patient weighed 3.6 kg (birth weight 3.7 kg) and was thin with significant skin folds. The abdomen was swollen, dense hepatomegaly of 6 cm and dense splenomegaly of about 4 cm were noted. In the inguinal region, enlarged lymph nodes were noted, and muscle activity was weak.

The initial hemoglobin level was 9.2 gm, 506,000 platelets, and 11,550 leukocytes. Urinalysis was normal, and bone marrow puncture revealed vacuolated lymphocytes and numerous foam cells. Chemical analysis of serum: total lipid level 834 mg / 100 ml, phospholipids 176 mg / 100 ml, triglycerides 141 mg / 100 ml, cholesterol 129 mg / 100 ml, bilirubin 0.3 mg / 100 ml, alkaline phosphatase 9.0% , СГОТ 90 units, СГПТ 50 units, cholinesterase 20 units, urea nitrogen 8.3 mg, fasting sugar 45 mg / 100 ml. A CT scan of the abdomen showed hepatosplenomegaly or bilaterally symmetrically enlarged adrenal glands with calcification.

A venous access port for dose administration was surgically implanted into the patient. After the port is connected to the ambulatory infusion apparatus, the patient is pre-administered with 1 mg / kg of diphenhydramine 20 minutes before the LAL infusion to counteract the potential anaphylaxis reactions caused by the infusion. Then the patient was injected with LKL at a dose of 1 mg / kg for 5 hours by intravenous infusion. The therapy was repeated once every 7 days for an indefinite period of time.

After two weeks of administering the first dose of LCL, the patient showed a significant improvement in weight gain and normalization of the size of the main organs of the abdominal cavity, as determined by ultrasound. Laboratory results show that LKL infusion restores lysosomal acid lipase activity in a patient and corrects associated abnormalities.

Example 19

Treatment of cholesterol ester accumulation disease (BNEC) by administration of recombinant LCL

A boy aged 3 years with an itchy abdominal rash was examined by his pediatrician. After examination of the abdomen, hepatomegaly was noted, which was confirmed by ultrasound. At this stage, the diagnosis was not made, and the patient underwent periodic monitoring.

At the age of 8, he was admitted to the hospital with gastroenteritis. Light microscopy of a liver biopsy revealed elevated levels of glycogen in the cytoplasm and small lipid drops in hepatocytes. Electron microscopy revealed membrane-bound lipid droplets with small electron-dense granules. A working diagnosis of type III glycogen accumulation disease (glycogenosis) was made, but the activity of skin fibroblasts was normal.

At the age of 10, hepatomegaly persisted and a second liver biopsy was taken. Light microscopy revealed an altered lobular structure of the liver parenchyma with distended hepatocytes containing cytoplasmic granules and vacuoles with slight periportal fibrosis. The activity of fibroblast acid lipase was 7% of the norm, which confirms the diagnosis of BNEC. Concentrations of total cholesterol (OX) in plasma, triglycerides (TG), low-density cholesterol lipoprotein (LDL-X) exceed 95% for a given age and gender and are 7.51, 3.24 and 5.58 mmol / l, respectively while the level of plasma high cholesterol lipoprotein (HDL-X) is below 5% at a level of 0.47 mmol / l; the patient was diagnosed with combined hyperlipidemia (hypercholesterolemia, hypertriglyceridemia, hypoalphalipoproteinemia and hyperbetalipoproteinemia).

A venous access port for dose administration was surgically implanted into the patient. After the port is connected to an ambulatory infusion apparatus, the patient is pre-administered with 5 mg / kg of diphenhydramine 20 minutes before the LCL infusion to counteract the potential anaphylaxis reactions caused by the infusion. Then the patient was injected with LKL at a dose of 5 mg / kg for 5 hours by intravenous infusion. The therapy was repeated once every 14 days for an indefinite period of time.

After two weeks of administering the first dose of LCL, the patient showed a significant improvement in weight gain and normalization of the size of the main organs, as determined by ultrasound. Laboratory results show that LKL infusion restores lysosomal acid lipase activity in a patient and corrects associated abnormalities.

Example 20

Description and composition of the drug

The LCL drug substance described herein (“SBC-102”) is a recombinant human lysosomal acid lipase (rhLCL) purified from egg white and obtained from transgenic Gallus birds. Excipients for SBC-102 are similar to those used for other products for the treatment of disorders of lysosomal accumulation (NLN), currently available on the market, and have been selected to maintain the stability of the drug.

SBC-102 is a clear, colorless, sterile liquid, supplied in a transparent type I borosilicate glass ampoule with an artificial latex (butyl) stopper coated with FluroTec® and an aluminum membrane. SBC-102 is supplied as an aqueous solution including SBC-102 (2 mg / ml), trisodium citrate dihydrate (13.7 mg / ml, USP), citric acid monohydrate (1.57 mg / ml, USP), human serum albumin (10 mg / ml, USP) and water for injection (to the final volume, USP). The pH value for SBC-102 is 5.9 ± 0.2. SBC-102 does not contain preservatives, and the ampoules are for single use only.

Each example in this application is provided to illustrate the invention, and not to limit the invention. It will be apparent to those skilled in the art that various modifications, combinations, additions, deletions, and variations can be made to the invention without reducing the scope and general idea of the invention. For example, the features illustrated or described as part of one embodiment of the invention may be used in another embodiment of the invention to provide another embodiment of the invention. The present invention covers such modifications, combinations, additions, deletions and variations.

All documents (e.g., US patents, US patent publications, publications) cited in this application are hereby incorporated by reference. Various modifications and variations of the present invention will become apparent to those skilled in the art without reducing the scope and principle of the invention.

While this invention has been shown separately and described with reference to examples of embodiments of the invention, those skilled in the art will understand that various changes in form and detail can be made without reducing the scope of the invention covered by the appended claims.

Claims (20)

1. A pharmaceutical composition for treating conditions caused by a lysosomal acid lipase deficiency, comprising isolated recombinant human lysosomal acid lipase (LKL), comprising SEQ ID NO: 2, and a pharmaceutically acceptable carrier, solvent or excipient, wherein the pharmaceutical composition is an aqueous solution which has a pH between about 5.6 and about 6.2, or a pH of 5.9 ± 0.2, and the LK is N-linked glycosylated at the position of Asn ¹⁵ , Asn ⁸⁰ , Asn ¹⁴⁰ , Asn ²⁵² and Asn ^{300 of the} sequence SEQ ID NO: 2 and includes the following N-linked glycosylation profile:
but. at position Asn ¹⁵ , GlcNAc4Man3GlcNAc2, or
Gal1GlcNAc4Man3GlcNAc2;
b. at position Asn ⁸⁰ , Phos2Man7GlcNAc2;
at. at position Asn ¹⁴⁰ , Phos1Man6GlcNAc2,
GlcNAc1Phos1Man6GlcNAc2;
Man3GlcNAc2;
GlcNAc2Man3GlcNAc2;
GlcNAc3Man3GlcNAc2;
GlcNAc4Man3G1cNAc2, or
Gal1GlcNAc4Man3GlcNAc2;
at position Asn ²⁵² , Man7GlcNAc2,
Man8GlcNAc2,
Man9GlcNAc2,
Phos1Man8GlcNAc2, or
Phos1Man9GlcNAc2; and
D. in position Asn ³⁰⁰ , GlcNAc2Man3GlcNAc2,
GlcNAc3Man3GlcNAc2,
GlcNAc4Man3GlcNAc2,
Gal1GlcNAc4Man3GlcNAc2,
GlcNAc5Man3GlcNAc2,
Gal1GlcNAc5Man3GlcNAc2,
GlcNAc6Man3GlcNAc2, or
Gal1GlcNAc6Man3GlcNAc2,
with Man = mannose,
GlcNAc = N-Acetylglucosamine
Phos = Phosphate
Gal = galactose. 2. The pharmaceutical composition according to p. 1, characterized in that the LCL comprises an N-glycan structure comprising phosphorylated mannose at the position of Asn ⁸⁰ , Asn ¹⁴⁰ or Asn ^{252 of the} sequence SEQ ID NO: 2. 3. The pharmaceutical composition according to claim 2, characterized in that the LCL comprises an N-glycan structure comprising phosphorylated mannose (M6P) at position Asn ¹⁴⁰ . 4. The pharmaceutical composition according to claim 1, characterized in that from about 10% to about 50% LAL in the composition includes N-glycan structures associated with Asn ¹⁴⁰ , which include M6P. 5. The pharmaceutical composition according to p. 2, characterized in that the LCL comprises an N-glycan structure comprising phosphorylated mannose (M6P) at position Asn ²⁵² . 6. The pharmaceutical composition according to claim 1, characterized in that at least 50% LAL in the composition includes N-glycan structures at position Asn ²⁵² , which include M6P. 7. The pharmaceutical composition according to claim 1, characterized in that the LCL comprises an N-glycan structure comprising terminal galactose at the position of Asn ¹⁵ , Asn ¹⁴⁰ or Asn ³⁰⁰ . 8. A pharmaceutical composition according to claim. 1, characterized in that from about 2% to about 10% LAL in the composition comprises N-glycan structures associated with Asn ^15, which include a terminal galactose. 9. The pharmaceutical composition according to claim 8, characterized in that at least 5% of the N-glycan structures associated with Asn ¹⁴⁰ contain terminal galactose. 10. A pharmaceutical composition according to claim. 9, characterized in that the terminal galactose LKL includes at position Asn ^300. 11. The pharmaceutical composition according to claim 1, characterized in that the position of Asn ^{51 is} non-glycosylated. 12. The pharmaceutical composition according to claim 1, characterized in that the LCL comprises xylose-free N-glycan structures, and less than 15% of the N-glycan structures contain sialic acid. 13. The pharmaceutical composition according to p. 12, wherein the LCL comprises xylose-free N-glycan structures, and less than 10% of the N-glycan structures contain sialic acid. 14. The pharmaceutical composition according to claim 13, wherein the LCL comprises xylose-free N-glycan structures, and less than 5% of the N-glycan structures contain sialic acid. 15. The pharmaceutical composition according to p. 1, characterized in that the LAL does not contain sialic acid or xylose. 16. The pharmaceutical composition according to p. 1, characterized in that the LKL does not contain fucose. 17. The pharmaceutical composition according to claim 1, characterized in that the LCL is obtained from the germ line of transgenic birds. 18. The pharmaceutical composition according to p. 17, wherein the LCL is obtained from the cells of the oviduct of the germ line of transgenic birds. 19. The pharmaceutical composition according to p. 17, wherein the bird is a chicken. 20. The pharmaceutical composition according to p. 1, characterized in that the composition includes at least one agent selected from the group consisting of trisodium citrate dehydrate, citric acid and human serum albumin.

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