Nucleic acid encoding ides

Abstract

The present invention includes nucleic acid encoding for an immunoglobulin G-degrading enzyme and method of producing the immunoglobulin G-degrading enzyme. Uses of the immunoglobulin G-degrading enzyme include research purposes, reducing a potential host immune response to an administered treatment and treating an autoimmune diseases or immune- mediated condition.

Description

Attorney Docket No.065830.11482 / 29WO1 NUCLEIC ACID ENCODING IDES CROSS REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to U.S. Provisional Application No.63 / 639,414, filed April 26, 2024, which is incorporated by reference herein in its entirety. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The contents of the electronic sequence listing (065830-29WO1.xml; size: 24,229 bytes; and date of creation: April 22, 2025) is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION

[0003] Immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) is an extracellular cysteine protease that can cleave human IgG in the lower hinge region with a high degree of specificity. Cleavage is a multi-step process providing F(ab’)2 and Fc fragments. (Wenig et al., PNAS.2004, 101(50):17371-17376, Pawel et al., EMBO Journal.2002, 21(7):1607-1615, and U.S. Patent No.7,666,582.)

[0004] IdeS has been proposed for a variety of different uses, based on its ability to cleave human IgG and decrease a host IgG immune response. Such uses include autoimmune disease treatment, transplantation, neutralizing anti-factor VIII (FVIII) antibodies during FVIII replacement therapy, treatment of vasculitis; and gene therapy involving viral vector gene delivery, where IdeS is used to decrease a subject’s IgG against the viral vector. (Björck, J. Clin. Cell. Immunol.2016, 7:2; U.S. Patent No.10,696,959; Bou-Jaoudeh et al., Haematologica.2023 Jan 19. doi: 10.3324 / haematol.2022.281895; Segelmark and Björck, Front. Immunol.2019, 10:2165, doi: 10.3389 / fimmu.2019.02165; International Patent Publication Nos. WO2020 / 016318, WO2022 / 266044 and WO2020 / 102740; Ros-Gañán et al., Clin. Transl. Immunology.2022, Feb 24;11(2):e1375, and Leborgne et al., Nat. Med.2020, 26(7):1096- 1101.)

[0005] Another example of the use of IdeS is the production of F(ab’)2. (U.S. Patent No. 11,053,280.)

[0006] Different functional IdeS variants containing one or more amino acid modifications and the ability to cleave human IgG have been identified. References mentioning variants include International Patent Publication Nos. WO2022 / 266044 and WO2020 / 016318; and U.S. Patent Nos.11,053,280, 10,696,959, and 7,666,582. 1 4930-2481-6953.1BRIEF SUMMARY OF THE INVENTION

[0007] The present invention features codon optimized nucleic acid encoding an immunoglobulin G-degrading enzyme (also referred to herein as an immunoglobulin G- degrading protein, immunoglobulin G-degrading enzyme of Streptococcus pyogenes, or IdeS) of SEQ ID NO: 9 or a derivative thereof. Uses of the encoded protein include reducing a potential host immune response to an administered treatment such as those involving a viral vector; tissue and organ transplantation; and treating an autoimmune diseases or immune-mediated condition.

[0008] Thus, a first describes a polynucleotide comprising a nucleic acid sequence encoding a protein comprising the sequence of SEQ ID NO: 9 or a variant of SEQ ID NO: 9 having 1, 2, 3, 4 or 5 N-terminal amino acids replaced with a signal peptide, wherein: (a) each codon encoding for asparagine in said protein is AAC; and (b) each codon encoding for serine in said protein is TCT.

[0009] A second aspect of the present invention describes a polynucleotide comprising a nucleic acid sequence encoding a protein comprising the sequence of SEQ ID NO: 9, wherein said nucleic acid sequence encoding said protein comprises in positions corresponding to SEQ ID NO: 1: a) codons 3, 5, 13, 22, 122, 137, 159, 172, 190, 195, 208, 224, 245, 257, 259, 262, 274, 281, 301, and 306 encoding for serine are each TCT; and b) codons 7, 33, 48, 59, 73, 82, 100, 102, 117, 154, 162, 203, 229, 233, 237, 246, 248, 260, 273, 289, 308, and 311 encoding for asparagine are each AAC.

[0010] A third aspect of the present invention describes a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 9, wherein the polynucleotide comprises a nucleic acid sequence at least 90% identical to any of SEQ ID NOs: 1-8.

[0011] In certain embodiments, the polynucleotide encoding a protein comprising the sequence of SEQ ID NO: 9 or variant thereof, is an expression cassette further comprises a promoter operatively coupled to the protein encoding sequence.

[0012] In certain embodiments, the promoter operatively coupled to the encoding nucleic sequence is isolated, part of a cellular chromosome and / or is present as an extrachromosomal element. Reference to “isolated” indicates not present in a cell. Examples of extrachromosomal elements include a plasmid, bacteriophage, and bacterial artificial chromosome.

[0013] Additional aspects include a recombinant prokaryotic cell comprising a polynucleotide described herein encoding a protein comprising the amino acid sequence of SEQ ID NO: 9 or variant thereof; the use of the recombinant cell to produce a protein comprising the amino acid sequence of SEQ ID NO: 9 or variant thereof; purifying the expressed protein; preparing a pharmaceutical composition using the purified protein; and methods of treatment comprising the pharmaceutical composition.2 4930-2481-6953.1

[0014] Other features and advantages of the present invention are apparent from additional descriptions provided herein, including different examples. The provided examples illustrate different components and methodology useful in practicing the present invention. Such examples do not limit the claimed invention. Based on the present disclosure, the skilled artisan can identify and employ other components and methodology useful for practicing the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIGs.1A, 1B and 1C illustrate a nucleic acid sequence encoding a mature IdeS of SEQ ID NO: 9, and further comprising an amino terminus methionine codon. The different codons are numbered in the figures. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention features codon optimized polynucleotides comprising an IdeS- encoding polynucleotide. Reference to “IdeS-encoding” provides for a sequence encoding a protein comprising SEQ ID NO: 9 or a variant thereof having 1, 2, 3, 4 or 5 N-terminal amino acids replaced with a signal peptide. Uses of the encoded protein include reducing a potential host immune response in a subject to an administered treatment such as those involving a viral vector or transplantation, and treating an autoimmune diseases or immune-mediated condition.

[0017] In certain embodiments, codon optimization helps provide for increased homogeneity of a protein comprising the amino acid sequence of SEQ ID NO: 9; and / or provides for increased titer and productivity. Without being limited to any particular theory, cellular protein expression can result in heterogeneity of an encoded protein, for example, due to codon wobble and amino acid misincorporation, which can be reduced by changing certain codons.

[0018] FIGs.1A, 1B and 1C illustrate a nucleic acid sequence encoding for the amino acid of SEQ ID NO: 9. SEQ ID NO: 9 comprises a mature IdeS and an added N-terminal methionine. The different codons are numbered in the figures.

[0019] Reference to “subject” indicates a mammal, including humans; non-human primates such as apes, gibbons, gorillas, chimpanzees, orangutans, macaques; domestic animals, such as dogs and cats; farm animals such as poultry and ducks, horses, cows, goats, sheep and pigs; and experimental animals such as mice, rats, rabbits, and guinea pigs. A preferred subject for treatment is a human subject. However, other subjects producing an IgG that can be cleaved by IdeS described herein can be treated.

[0020] Reference to an expression cassette indicates a polynucleotide sequence encoding a protein operatively linked to one or more regulatory element providing for protein expression.3 4930-2481-6953.1One type of regulatory element is a promoter, which binds RNA polymerase and the necessary transcription factors to initiate transcription. When encoding protein, the produced RNA sequence will also encode a termination sequence at the end of the coding sequence and the expression cassette will further comprise regulatory elements. Additional regulatory elements include those impacting RNA expression, RNA stability, and / or protein production.

[0021] DNA vectors may be single-stranded, double-stranded, or contain a combination of single and double stranded regions. The vector may also include more than transgene and multiple regulatory elements of the same or different types.

[0022] The term “operatively linked” refers to the association of two or more nucleic acid segments on a single nucleic acid where the function or processing of one is affected by the other. For example, a promoter operatively linked to a sequence encoding a protein facilitates DNA transcription producing RNA.

[0023] The singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

[0024] As used herein, the conjunctive term “and / or” between multiple recited elements is understood to encompass both individual and combined options. For instance, where two elements are conjoined by “and / or”, a first option refers to the applicability of the first option without the second, a second option refers to the applicability of the second option without the first, and a third option refers to the applicability of the first and second options together. Similarly, three elements are conjoined by “and / or”, refers to each element individually, the combination of any two of elements, or all three elements. Any one of the options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or”. Concurrent applicability of each possible element combination or all options is understood to fall within the meaning of the term “and / or.”

[0025] Reference to terms such as “including”, “for example”, “e.g.,”, “such as” followed by different members or examples, are open-ended descriptions where the listed members or examples are illustrative and other members or examples can be provided or used.

[0026] The terms “polypeptides,” “proteins” and “peptides” can be used interchangeably to refer to an amino acid sequence without regard to function. Polypeptides and peptides contain at least two amino acids, while proteins contain at least about 10 amino acid acids. The provided amino acids include naturally occurring amino acids and amino acids provided by cellular modification.

[0027] Reference to “comprise”, and variations such as “comprises” and “comprising”, used with respect to an element or group of elements is open-ended and does not exclude additional unrecited elements or method steps. Terms such as “including”, “containing” and “characterized4 4930-2481-6953.1by” are synonymous with comprising. In the different aspects and embodiments described herein reference to an open-ended term such as “comprising” can be replaced by the terms “consisting” or “consisting essentially of”.

[0028] Reference to “consisting of” excludes any element, step, or ingredient not specified in the listed claim elements, where such element, step or ingredient is related to the claimed invention.

[0029] Reference to “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.

[0030] The term “about” refers to a value within 10% of the underlying parameter (i.e., plus or minus 10%). For example, “about 1:10” includes 1.1:10.1 or 0.9:9.9, and “about 5 hours” includes 4.5 hours or 5.5 hours. The term “about” at the beginning of a string of values modifies each of the values by 10%. In a further embodiment, “about” provides for a value with 5% of the underlying parameter.

[0031] All numerical values or numerical ranges include integers within such ranges and fractions of the values or the integers within ranges unless the context clearly indicates otherwise. Thus, to illustrate, reference to reduction of 95% or more includes 95%, 96%, 97%, 98%, 99%, 100%, as well as 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, etc., 96.1%, 96.2%, 96.3%, 96.4%, 96.5% and so forth; reference to a numerical range, such as “1-4” includes 2, 3, as well as 1.1, 1.2, 1.3, 1.4 and so forth; reference to “1 to 4 weeks” includes 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days; reference to a numerical range, such as “0.01 to 10” includes 0.011, 0.012, 0.013 and so forth, as well as 9.5, 9.6, 9.7, 9.8, 9.9 and 10 and so forth. For example, a dosage of “0.01 mg / kg to 10 mg / kg” body weight of a subject includes 0.011 mg / kg, 0.012 mg / kg, 0.013 mg / kg, 0.014 mg / kg, 0.015 mg / kg and so forth as well as 9.5 mg / kg, 9.6 mg / kg, 9.7 mg / kg, 9.8 mg / kg, 9.9 mg / kg and so forth.

[0032] Reference to an integer with more (greater) or less than includes numbers greater or less than the reference number, respectively. Thus, for example, reference to more than 2 includes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15; and administration “two or more” times includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15, or more times.

[0033] Reference to an indicated percent identity to one or more reference sequences, and similar language throughout the specification providing for an indicated percent identity to one or more reference sequences, provides the indicated percent identity or percent identity range independently to each of the referenced sequences.

[0034] Reference to a percent “identical”, “identity” and similar terminology are with respect to two sequences having maximal alignment in a particular area. The provided area is with respect to the indicated reference sequence. For example, sequence “identical” or “identity” to a5 4930-2481-6953.1polypeptide of SEQ ID NO: 9 can be calculated by determining the number of identical amino acids in aligned sequences, dividing by the total number of amino acids in SEQ ID NO: 9 and multiplying by 100. Percent “identical” or “identity” for nucleic acid sequences can be determined in an analogous manner where nucleotides to the reference sequence are aligned to achieve maximal alignment taking into account nucleotide differences and gaps, dividing by the total number of nucleotides in the reference sequence and multiplying by 100.

[0035] Reference to differing with respect to a reference sequence (e.g., amino acid or nucleic acid sequence), provides for the indicated number of differences, where each difference is selected from a substitution, deletion, and addition. In certain embodiments for an amino acid sequence, each difference is a substitution. In a further embodiment each substitution is a conservative substitution.

[0036] Various references including articles and patent publications are cited or described in the background and throughout the specification. Each of these references is herein incorporated by reference in their entirety. None of the references are admitted to be prior art with respect to any inventions disclosed or claimed. In some cases, particular references are indicated to be incorporated by reference herein to highlight the incorporation.

[0037] The definitions provided herein, including those in the present section and other sections of the application apply throughout the present application.

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning commonly understood to one of ordinary skill in the art to which this invention pertains.

[0039] The description has been separated into various sections and paragraphs, and provides various embodiments. These separations should not be considered as disconnecting the substance of a paragraph or section or embodiment from the substance of another paragraph or section or embodiment. The provided descriptions have broad application and encompass all the combinations of the various sections, paragraphs and sentences that can be contemplated. The discussion of any embodiment is meant only to be exemplary and is not intended to suggest the scope of the disclosure, including the claims (unless otherwise provided in the claims), is limited to these examples. I. Immunoglobulin G-degrading Enzyme

[0040] Naturally occurring IdeS is produced as a full-length protein comprising a secretory sequence, which is cleaved to a mature sequence. The amino acid sequence of SEQ ID NO: 9, provides the mature IdeS sequence further comprising an amino terminal methionine.6 4930-2481-6953.1

[0041] In certain embodiments, a signal peptide is fused to the amino terminus of SEQ ID NO: 9, or one or more amino terminal amino acids (e.g., 1, 2, 3, 4 or 5) starting with methionine is replaced with a signal peptide providing for periplasmic secretion. Examples of signal peptides providing for periplasmic secretion include PelB, OmpA, SpA, PhoA, LamB, and DsbA. (Pouresmaeil and Azizi-Dargahlou Arch Microbiol 205, 212 (2023), and Zhang, et al., Microb Cell Fact 17, 50 (2018).)

[0042] In certain embodiments the immunoglobulin G-degrading enzyme further comprises an affinity tag that can be used, for example, to facilitate purification. The affinity tag can be placed at the amino or carboxy terminus. In a further embodiment, a protease cleavage tag can be used to facilitate removing an infinity tag. An example of an affinity tag is a His-tag. An example of a protease cleavage site is a Tobacco Etch Virus (TEV) cleavage cite which can be used in combination with a TEV protease. II. IdeS-Encoding Polynucleotides

[0043] The IdeS-encoding polynucleotides described herein can be used to express an immunoglobulin G-degrading enzyme. Due to the degeneracy of the genetic code, a large number of nucleic acid sequences can encode for the immunoglobulin G-degrading enzyme sequence provided by SEQ ID NO: 9. In some cases, a particular codon can affect the level of expression and increase the possibility of incorrect amino acid incorporation. (See, for example, Siddhartha et al, DNA Research, Volume 23, Issue 5, October 2016, pages 441–449, and Zhang et al., Biochemistry.2013 Nov 12;52(45):8165-76, both of which are hereby incorporated by reference herein in their entirety.

[0044] In certain embodiments the polynucleotide encodes for an amino acid sequence comprising the sequence of SEQ ID NO: 9 or a variant of SEQ ID NO: 9 having 1, 2, 3, 4 or 5 N-terminal amino acids replaced with a signal peptide, wherein: (a) each codon encoding for asparagine in the protein is AAC; and (b) each codon encoding for serine in the protein is TCT.

[0045] In certain embodiments, the polynucleotide encodes an amino acid sequence of SEQ ID NO: 9 or variant thereof, wherein the encoding sequence comprises any one, two, or all three of (a) each codon encoding for proline in the protein is CCG; (b) each codon encoding for threonine in the protein is ACT; and (c) each codon encoding for histidine in said protein is CAC.

[0046] In certain embodiments the polynucleotide encodes for an amino acid sequence comprising the sequence of SEQ ID NO: 9 or a variant of SEQ ID NO: 9 having 1, 2, 3, 4 or 5 N-terminal amino acids replaced with a signal peptide wherein:7 4930-2481-6953.1a) codons 3, 5, 13, 22, 122, 137, 159, 172, 190, 195, 208, 224, 245, 257, 259, 262, 274, 281, 301, and 306 encoding for serine are each TCT; and b) codons 7, 33, 48, 59, 73, 82, 100, 102, 117, 154, 162, 203, 229, 233, 237, 246, 248, 260, 273, 289, 308 and 311 encoding for asparagine are each AAC.

[0047] The codon numbering is provided with respect to the numbering provided in FIGs.1A, 1B and 1C.

[0048] In certain embodiments, the polynucleotide encodes an amino acid sequence of SEQ ID NO: 9 or a variant thereof, wherein the nucleic acid sequence encoding the protein comprises in positions corresponding to SEQ ID NO: 1 any one, two, or all three of: (a) codons 17, 30, 31, 44, 94, 134, 145, 165, 167, and 175, encoding for proline are each CCG; (b) codons 16, 21, 25, 29, 35, 55, 57, 70, 115, 138, 161, 166, 185, 194, 216, 226, 255, 299, 302, and 310 encoding for threonine are each ACT; and (c) codons 19, 42, 76, 93, 118, 140, 147, 163, 197, 225, and 234 encoding for histidine are each CAC.

[0049] In further embodiments, the polynucleotide encoding the amino acid sequence comprising the sequence of SEQ ID NO: 9 comprises a sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the nucleic acid sequence of SEQ D NO: 3, differs from SEQ ID NO: 3 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides, or comprises the sequence of SEQ ID NO: 3.

[0050] In certain embodiments, the polynucleotide encoding the amino acid sequence of SEQ ID NO: 9 comprises a sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the nucleic acid sequence of any of SEQ ID NOs: 1-8, differs from any of SEQ ID NOs: 1-8 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides, or comprises the sequence of any of SEQ ID NOs: 1-8. III. Recombinant Expression

[0051] In certain embodiments an immunoglobulin G-degrading enzyme is recombinantly expressed in a prokaryotic host using an expression cassette comprising IdeS-encoding nucleic acid. The expression cassette provides regulatory and expression control elements needed for RNA production, processing and translation.

[0052] Recombinant expression can be obtained from encoding nucleic acid present in host chromosome and / or present as extrachromosomal elements. Reference to “recombinant” refers to one or more components not occurring in nature and / or a combination of components not occurring in nature. The optimized polynucleotide sequences described herein differ from naturally occurring sequence and may also be combined with different elements such as a heterologous promoter and other regulatory or expression control elements. In certain8 4930-2481-6953.1embodiments, nucleic acid regulatory and expression control elements are provided affecting nucleic acid processing, transcription, and / or translation.

[0053] A promoter is a DNA region providing for transcription. In general, transcribed nucleic acid is located 3’ of a promoter sequence. In certain embodiments, a promoter sequence is coupled to an enhancer. Enhancers are DNA regions that increase promoter transcription. Enhancers can be adjacent or inside a promoter or can be distal. Typically, enhancers are located upstream of a promoter, but can be located downstream or within a promoter sequence.

[0054] A variety of different promoters can be used for prokaryotic expression including constitutive promotors, inducible promoters, and stationary phase promoter. Examples of prokaryotic promoters include T3, T7, T7lac, rpos, mms, trc, tetA, tac, trc, lac, trp, phage promoter pL, rhaPBAD, proU, recA, tacM, PL, araPBAD, cspA, cspB, phyL, NBP3510, P43, Psac, P170, Pgrac, SP6 and synthetic promoters. (See, for example, Duzenli and Okay, AIMS Bioengineering, 2020, volume 7, issue 2: 62-81; Carrillo Rincón, Microbial Biotechnology, 2023, 16, 961–976; and Pouresmaeil and Azizi-Dargahlou, Arch Microbiol 205, 212 (2023); each of which is hereby incorporated by reference herein in its entirety.) In certain embodiments, the promoter is a lac promoter, and isopropyl-β-D-thiogalactoside (IPTG) is used for induction.

[0055] In certain embodiments, the polynucleotide comprises a Shine-Dalgarno sequence providing a ribosomal binding site and facilitating RNA translation. (See, for example, Wen et al., RNA Biol.2021 Nov;18(11):1489-1500, hereby incorporated by reference herein in its entirety.)

[0056] In certain embodiments, a polyadenylation signal is present. The polyadenylation signal facilitates addition of a polyA to transcribed RNA. Prokaryotic polyadenylation may be involved in RNA metabolism, sch as mRNA turnover. (Hajnsdorf and Kaberdin, 2018 Phil. Trans. R. Soc. B 373: 20180166, hereby incorporated by reference herein in its entirety.)

[0057] Plasmids can be used as extrachromosomal elements and / or for insertion of DNA into a host chromosomal. In addition to an expression cassette comprising IdeS-encoding polynucleotide, a plasmid comprises additional elements providing replication such as an origin of replication, and a selectable marker.

[0058] A selectable marker provides for a growth advantage in the presence of certain agents or use of certain media, allowing for selection of cells containing the marker. Examples of selectable markers include antibiotic resistance genes, and enhanced ability to utilize certain nutrients such as particular amino acids.

[0059] In certain embodiments the IdeS-encoding polynucleotide is present in a bacteriophage nucleic acid or a bacterial artificial chromosomal. The bacterial artificial chromosomal9 4930-2481-6953.1comprises elements needed for replication and cell maintenance. The bacteriophage nucleic acid comprises elements for packaging into a bacteriophage.

[0060] In certain embodiments, the prokaryotic host is bacteria. In further embodiments, the host is Escherichia coli, Bacillus spp., Bacillus subtilis, Lactococcus lactis, Lactobacillus acidophilus, Corynebacterium glutamicum, Streptomyces spp., Streptomyces lividans, Pseudomonas spp., Pseudomonas fluorescens, Pichia pastoris, Streptococcus spp., Streptococcus pyogenes, or Cyanobacteria diphtheria.

[0061] Prokaryotes can grow in a variety of different media. Culture media components can include nutrients, energy sources, and other substances providing for growth. The culture media can be a defined media or a complex media. In certain embodiments, the energy source is glycerol and / or glucose.

[0062] Recombinant protein production and purification, in general, is described in Wingfield Curr Protoc Protein Sci.2015 Apr 1;80:6.1.1-6.1.35, hereby incorporated by reference herein in its entirety. Examples of techniques used for recombination expression and purification of immunoglobulin degrading enzymes includes those provided herein, in International Patent Publication Nos. WO2022 / 266044 and WO2020 / 016318, and in U.S. Patent Nos.10,696,959 and 7,666,582, each of which are incorporated by reference herein in its entirety,

[0063] In certain embodiments, recombinant protein production produces a protein comprising or consisting of SEQ ID NO: 9 or a SEQ ID NO: 9 deletion variant lacking the N-terminal methionine.

[0064] Preferably, the IdeS-encoding nucleic acid provides increased homogeneity of the encoded sequence when expressed in a prokaryote. Increased homogeneity can, for example, facilitate purification of protein having the correct amino acid sequence.

[0065] Reference to “purifying” or “purified” protein from a cell indicates separation from one or more cellular components such as host proteins. In different embodiments, the protein is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, or at least 97% purified with respect to recombinant cell host protein.

[0066] In the certain embodiments, immunoglobulin G-degrading enzyme purity using reverse phase liquid chromatography is at least 85%, at least 90%, or at least 95%.

[0067] In the certain embodiments, immunoglobulin G-degrading enzyme purity using size exclusion chromatography is at least 94%, at least 95%, at least 96%, at least 97% or at least 98%.

[0068] In the certain embodiments, immunoglobulin G-degrading enzyme purity using CE-SEC reduced chromatography is at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95%.10 4930-2481-6953.1IV. Gene Therapy

[0069] In certain embodiments, recombinantly expressed immunoglobulin G-degrading protein is used in conjunction with gene therapy. The protein can be used, for example, to inhibit an immune response against a vector used in gene therapy and / or an immune response directed to a gene therapy product.

[0070] Gene therapy includes both loss-of-function and gain-of-function. The term “loss-of- function” in reference to a genetic defect, refers to a mutation in a gene in which the protein encoded by the gene exhibits either a partial or a full loss of function that is normally associated with the wild-type protein. The term “gain-of-function” in reference to a genetic defect refers to a mutation in a gene in which the protein encoded by the gene acquires a function not normally associated with the wild type protein causes or contributes to a disease or disorder. The gain-of- function mutation can be a deletion, addition, or substitution of a nucleotide or nucleotides in the gene, giving rise to a change in the encoded protein function. In certain embodiments, the gain- of-function mutation changes the function of the mutant protein or causes interactions with other proteins. In certain embodiments, the gain-of-function mutation causes a decrease in or removal of normal wild-type protein, for example, by interaction of the altered mutant protein with the normal wild-type protein.

[0071] A transgene can be used to provide a protein having a desired activity and / or biologically active nucleic acid. In different embodiments, the transgene encodes a viral antigen, a bacterial antigen, a therapeutic protein, a short hair pin RNA (shRNA), a small interfering RNA (siRNA), a microRNA (miRNA), a ribozyme, an antisense RNA, a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 construct, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a small activating RNA (saRNA). IV.A. Therapeutic Proteins

[0072] In certain embodiments the transgene encodes a protein having a desired activity. Examples of transgenes include those providing a healthy copy of gene in a subject where the subject gene is defective, a modified gene that can help treat a disease or disorder, or a new gene encoding a protein providing a beneficial effect.

[0073] In different embodiments, the transgene encodes GAA (acid alpha-glucosidase) for treatment of Pompe disease; TPP1 (tripeptidyl peptidase-1) for treatment of late infantile neuronal ceroid lipofuscinosis type 2 (CLN2); ATP7B (copper transporting ATPase2) for treatment of Wilson’s disease; alpha galactosidase for treatment of Fabry disease; ASS1 (arginosuccinate synthase) for treatment of Citrullinemia Type 1; beta-glucocerebrosidase for treatment of Gaucher disease Type 1; beta-hexosaminidase A for treatment of Tay-Sachs disease; SERPING1 (C1 protease inhibitor or C1 esterase inhibitor) for treatment of hereditary11 4930-2481-6953.1angioedema (HAE), also known as C1 inhibitor deficiency type I and type II); or glucose-6- phosphatase for treatment of glycogen storage disease type I (GSDI).

[0074] In different embodiments, the transgene encodes insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor α (TGFα), platelet-derived growth factor (PDGF), insulin growth factors I or II (IGF-I or IGF-II), TGFβ, activin, bone morphogenic protein (BMP), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 or NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line derived neurotrophic factor (GDNF), neurturin, agrin, netrin-1 or netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog or tyrosine hydroxylase.

[0075] In different embodiments, the transgene encodes thrombopoietin (TPO), an interleukin (IL-1 through IL-36), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factors α or β, interferons α, β, or γ, stem cell factor, flk-2 / flt3 ligand, IgG, IgM, IgA, IgD or IgE, chimeric immunoglobulins, an antibody, humanized antibody, single chain antibody, T cell receptor, chimeric T cell receptor, single chain T cell receptor, class I or class II MHC molecules. Antibodies and immunoglobulins can, for example, can target cancer cells or other disease or disorder causing cells.

[0076] In different embodiments, the transgene encodes CFTR (cystic fibrosis transmembrane regulator protein), a blood coagulation (clotting) factor (Factor XIII, Factor IX (FIX), Factor VIII (FVIII), Factor X, Factor VII, Factor VIIa, or protein C) a gain of function blood coagulation factor, erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, β-globin, α-globin, spectrin, α-antitrypsin, adenosine deaminase (ADA), a metal transporter (ATP7A or ATP7), sulfamidase, an enzyme involved in lysosomal storage disease (ARSA), hypoxanthine guanine phosphoribosyl transferase, β-25 glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain keto acid dehydrogenase, a hormone, a growth factor, insulin-like growth factor 1 or 2, platelet derived growth factor, epidermal growth factor, nerve growth factor, neurotrophic factor -3 and -4, brain-derived neurotrophic factor, glial derived growth factor, transforming growth factor α and β, a cytokine, α-interferon, β-interferon, interferon-γ, interleukin-2, interleukin-4, interleukin 12, granulocyte-macrophage colony stimulating factor, lymphotoxin, a suicide gene product, herpes simplex virus thymidine kinase,12 4930-2481-6953.1cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, tumor necrosis factor, a drug resistance protein, a tumor suppressor protein (e.g., p53, Rb, Wt-1, NF1, Von Hippel–Lindau (VHL), and adenomatous polyposis coli (APC)), a peptide with immunomodulatory properties, a tolerogenic or immunogenic peptide or protein Tregitope or hCDR1, insulin, glucokinase, guanylate cyclase 2D (LCA-GUCY2D), retinal pigment epithelium-specific 65 kDa protein (RPE65), Rab escort protein 1 (choroideremia), LCA 5 (LCA-lebercilin), ornithine ketoacid aminotransferase (gyrate atrophy), retinoschisin 1 (X-linked retinoschisis), X-linked retinitis pigmentosa GTPase (XLRP), MER proto-oncogene tyrosine kinase (MERTK) (autosomal recessive (AR) forms of retinitis pigmentosa (RP)), ABCA4 (Stargardt), ACHM 2, 3 and 4 (achromatopsia), an anti-vascular endothelial growth factor (VEGF) agent polypeptide (e.g., bevacizumab, brolucizumab, ranibizumab, aflibercept), DFNB1 (connexin 26 deafness), USH1C (Usher’s syndrome 1C), PKD-1 or PKD-2 (polycystic kidney disease), TPP1 (tripeptidyl peptidase-1), a sulfatase, N-acetylglucosamine-1-phosphate transferase, cathepsin A, GM2-AP, NPC1, VPC2, a sphingolipid activator protein, or one or more donor sequences used as repair templates for genome editing.

[0077] In different embodiments, the transgene construct encodes erythropoietin (EPO) for treatment of anemia; interferon-alpha, interferon-beta, or interferon-gamma for treatment of various immune disorders, viral infections and cancer; an interleukin (IL), including any one of IL-1 through IL-36, and corresponding receptors, for treatment of various inflammatory diseases or immuno-deficiencies; a chemokine, including chemokine (C-X-C motif) ligand 5 (CXCL5) for treatment of immune disorders; granulocyte-colony stimulating factor (G-CSF) for treatment of immune disorders such as Crohn’s disease; granulocyte-macrophage colony stimulating factor (GM-CSF) for treatment of various human inflammatory diseases; macrophage colony stimulating factor (M-CSF) for treatment of various human inflammatory diseases; keratinocyte growth factor (KGF) for treatment of epithelial tissue damage; chemokines such as monocyte chemoattractant protein-1 (MCP-1) for treatment of recurrent miscarriage, HIV-related complications, and insulin resistance; tumor necrosis factor (TNF) and receptors for treatment of various immune disorders; alpha1-antitrypsin for treatment of emphysema or chronic obstructive pulmonary disease (COPD); alpha-L-iduronidase for treatment of mucopolysaccharidosis I (MPS I); ornithine transcarbamoylase (OTC) for treatment of OTC deficiency; phenylalanine hydroxylase (PAH) or phenylalanine ammonia-lyase (PAL) for treatment of phenylketonuria (PKU); lipoprotein lipase for treatment of lipoprotein lipase deficiency; apolipoprotein for treatment of apolipoprotein (Apo) A-I deficiency; low-density lipoprotein receptor (LDL-R) for treatment of familial hypercholesterolemia (FH); albumin for treatment of hypoalbuminemia; lecithin cholesterol acyltransferase (LCAT); carbamoyl synthetase I; argininosuccinate13 4930-2481-6953.1synthetase; argininosuccinate lyase; arginase; fumarylacetoacetate hydrolase; porphobilinogen deaminase; cystathionine beta-synthase for treatment of homocystinuria; branched chain ketoacid decarboxylase; isovaleryl-CoA dehydrogenase; propionyl CoA carboxylase; methylmalonyl-CoA mutase; glutaryl CoA dehydrogenase; insulin; pyruvate carboxylase; hepatic phosphorylase; phosphorylase kinase; glycine decarboxylase; H-protein; T-protein; cystic fibrosis transmembrane regulator (CFTR); ATP-binding cassette, sub-family A (ABC1), member 4 (ABCA4) for the treatment of Stargardt disease; or dystrophin.

[0078] In further embodiments the transgene encodes a protein for treating a disease or disorder selected from the group consisting of: hereditary angioedema, Pompe disease, hemophilia A, hemophilia B, Fabry disease, wet macular degeneration, Leber hereditary optic neuropathy, and Stargardt disease. IV.B. Biologically Active Nucleic Acid

[0079] Biologically activity nucleic acid can alter the activity of other nucleic acid. In certain embodiments, an immunoglobulin G-degrading enzyme is used in conjunction with gene therapy comprising biologically active nucleic acid. In certain embodiments, the biologically active nucleic acid is inhibitory nucleic acid. Inhibitory nucleic acid inhibit the activity of a nucleic acid, such as nucleic acid involved in transcription or translation. Examples of inhibitory nucleic acid include a short hairpin RNA (shRNA), a small interfering RNA (siRNA), a microRNA (miRNA), a ribozyme, and an antisense RNA. In certain embodiments, the biologically activity nucleic acid is small activating RNA (saRNA), which can increase transcription. (Tan et al., Gene Transcription. Molecules.2021 Oct 28;26(21):6530, hereby incorporated by reference herein in its entirety.)

[0080] In different embodiments, the inhibitory nucleic acid binds to a gene, a transcript of a gene, or a transcript of a gene associated with a polynucleotide repeat disease, a huntingtin (HTT) gene, a gene associated with dentatorubropallidoluysian atrophy (atrophin 1, ATN1), androgen receptor on the X chromosome in spinobulbar muscular atrophy, human Ataxin-1, -2, -3, and -7, Cav2.1 P / Q voltage-dependent calcium channel (CACNA1A), TATA-binding protein, Ataxin 8 opposite strand (ATXN8OS), serine / threonine-protein phosphatase 2A 55 kDa regulatory subunit B beta isoform in spinocerebellar ataxia (type 1, 2, 3, 6, 7, 8, 1217), FMR1 (fragile X mental retardation 1) in fragile X syndrome, FMR1 (fragile X mental retardation 1) in fragile X-associated tremor / ataxia syndrome, FMR2 (fragile X mental retardation 2) or AF4 / FMR2 family member 2 in fragile XE mental retardation; myotonin-protein kinase (MT- PK) in myotonic dystrophy; Frataxin in Friedreich’s ataxia; a mutant of superoxide dismutase 1 (SOD1) gene in amyotrophic lateral sclerosis; a gene involved in pathogenesis of Parkinson’s disease and / or Alzheimer’s disease; apolipoprotein B (APOB), proprotein convertase14 4930-2481-6953.1subtilisin / kexin type 9 (PCSK9), for hypercholesterolemia; HIV Tat, human immunodeficiency virus transactivator of transcription gene, in HIV infection; HIV TAR, HIV TAR, human immunodeficiency virus transactivator response element gene, in HIV infection; C-C chemokine receptor (CCR5) in HIV infection; Rous sarcoma virus (RSV) nucleocapsid protein in RSV infection; liver-specific microRNA (miR-122) in hepatitis C virus infection; p53, acute kidney injury or delayed graft function kidney transplant or kidney injury acute renal failure; protein kinase N3 (PKN3) in advance recurrent or metastatic solid malignancies; LMP2 also known as proteasome subunit beta-type 9 (PSMB 9), metastatic melanoma; LMP7, also known as proteasome subunit beta-type 8 (PSMB 8), metastatic melanoma; MECL1 also known as proteasome subunit beta-type 10 (PSMB 10), metastatic melanoma; vascular endothelial growth factor (VEGF) in solid tumors; kinesin spindle protein in solid tumors, apoptosis suppressor B- cell CLL / lymphoma (BCL-2) in chronic myeloid leukemia; ribonucleotide reductase M2 (RRM2) in solid tumors; Furin in solid tumors; polo-like kinase 1 (PLK1) in liver tumors, diacylglycerol acyltransferase 1 (DGAT1) in hepatitis C infection; beta-catenin in familial adenomatous polyposis; beta2 adrenergic receptor, glaucoma; RTP801 / Redd1 also known as DNA damage-inducible transcript 4 protein, in diabetic macular edema (DME) or age-related macular degeneration; vascular endothelial growth factor receptor I (VEGFR1) in age-related macular degeneration or choroidal neovascularization; caspase 2 in non-arteritic ischaemic optic neuropathy; keratin 6A N17K mutant protein in pachyonychia congenital; influenza A virus genome / gene sequences in influenza infection; severe acute respiratory syndrome (SARS) coronavirus genome / gene sequences in SARS infection; respiratory syncytial virus genome / gene sequences in respiratory syncytial virus infection; Ebola filovirus genome / gene sequence in Ebola infection; hepatitis B and C virus genome / gene sequences in hepatitis B and C infection; herpes simplex virus (HSV) genome / gene sequences in HSV infection; coxsackievirus B3 genome / gene sequences in coxsackievirus B3 infection; silencing of a pathogenic allele of a gene (allele-specific silencing) like torsin A (TOR1A) in primary dystonia, pan-class I and HLA-allele specific in transplant; and mutant rhodopsin gene (RHO) in autosomal dominantly inherited retinitis pigmentosa (adRP). IV.C. Gene Editing

[0081] In certain embodiments, an immunoglobulin G-degrading enzyme is used in conjunction with gene editing nucleic acid or enzymes. Examples of gene editing nucleic acid and enzymes include ZFN, TALEN, and CRISPR-Cas9. In different embodiments gene editing edits a subject’s DNA to provide a therapeutic protein as provided in Section IV.A. supra., or disrupt a gene as provided in Section IV.B. supra.15 4930-2481-6953.1V. Viral Vectors

[0082] A viral vector comprises recombinant viral nucleic acid encapsidated in a protein capsid. The viral vector can deliver the nucleic acid to cells or tissues. Depending on the particular vector, the viral vector may further comprise a viral envelope. Examples of viral vectors that can be used for gene delivery include, adenovirus vectors, recombinant adeno-associated virus vector, retrovirus vectors and herpes simplex vectors.

[0083] Different serotypes exist within different types of viruses. The different serotypes can provide for different activities, such as cell or tissue tropism and likelihood of generating a host immune response. The term “serotype” broadly refers to both serologically distinct viruses as well as viruses not serologically distinct that can be within a subgroup or a variant of a given serotype. Serologic distinctiveness can be determined based on the lack of cross-reactivity between antibodies to one capsid as compared to another capsid. Such cross-reactivity differences are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to VP1, VP2, and / or VP3 sequence differences of AAV serotypes).

[0084] As more naturally occurring virus isolates are discovered or capsid mutants generated, there may or may not be serological differences with any of the currently existing serotypes. Thus, in cases where the new virus has no serological difference, this new virus would be a subgroup or variant of the corresponding serotype. V.A. Adenovirus Vectors

[0085] Adenoviruses are non-enveloped double-stranded DNA viruses. Recombinant adenovirus vectors comprise recombinant adenovirus nucleic acid lacking one or more protein involved in viral replication, and further comprise an adenoviral capsid. Recombinant adenovirus vectors can be produced containing different amounts of adenoviral DNA. The adenovirus (Ad) genome is flanked by hairpin-like inverted terminal repeats (ITRs) varying in length from 30–371 bp at its termini. The ITRs serve as self-priming structures that promote primase-independent DNA replication. A packaging signal located at the left arm of the genome is required for viral genome packaging. (Liu and Seol (2020) BMB Reports; 53(11):565-575; and Bulcha et al., (2021) Sig. Transduct. Target Ther.6:53.)

[0086] In certain embodiments, the recombinant adenovirus vector is a third-generation vector, which are also referred to as “gutless” or “helper-dependent”. Gutless vectors can be produced from recombinant adenovirus nucleic acid where all, or substantially all viral sequences, except for the ITRs and the packaging signal, are not present. Gutless adenovirus vectors are high capacity vectors able to accommodate up to about 36 kb of DNA insert. Preferred recombinant adenovirus nucleic acid is about 27 kb to about 37 kb. Stuffer sequences can be added to recombinant adenovirus nucleic acid to increase nucleic acid size and capsid incorporation.16 4930-2481-6953.1Preferred stuffer sequences avoid coding sequences, repetitive sequences, recombination sequences, and immunogenic sequences. (Liu and Seol (2020) BMB Reports, 53(11):565-575; Bulcha et al., (2021) Sig. Transduct. Target Ther.6:53; and Sandig et al., PNAS (2000) 97(3):1002-1007; each of which are hereby incorporated by reference herein in their entirety.)

[0087] In certain embodiments, recombinant adenovirus vectors can be produced based on rare human serotypes or chimpanzee serotypes. The use of chimpanzee and rare human serotypes may be helpful in reducing host immune response against recombinant adenovirus vectors due to preexisting immunity. (Guo et al., (2018) Human vaccines & immunotherapeutics, 14(7):1679– 1685 and Bulcha et al., (2021) Sig. Transduct. Target Ther.6:53.)

[0088] Adenovirus vectors can be produced by supplying viral proteins needed for vector production in trans using, for example, appropriate helper viruses or plasmids and cell lines. (Liu and Seol (2020) BMB Reports; 53(11):565-575; and Bulcha et al., (2021) Sig. Transduct. Target Ther.6:53.) V.B. Recombinant AAV Vectors

[0089] Recombinant adeno-associated viral (also referred to herein as “rAAV”) vectors are based on the adeno-associated virus. The adeno-associated virus is a single-strand DNA virus containing a 4.7-kb genome flanked by 145-nt ITRs on both ends of the genome. ITR activity is important for self-priming and packaging, and may also provide additional activity such as promoter activity. AAV 5’ and 3’ ITRs can vary in size and the 5’ and 3’ inverted repeats need not be exact inverted repeats.

[0090] A rAAV vector contains AAV recombinant nucleic acid and a viral capsid. The rAAV recombinant nucleic acid lacks one or more AAV protein encoding sequence involved in viral replication. In certain embodiments, the rAAV vector contains an AAV 5’ and / or 3’ ITR along with a DNA insert. In certain embodiments, rAAV nucleic acid comprise a 5’ ITR and / or 3’ ITR independently selected from 5’ and 3’ ITRs provided in AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.74 and AAV3B ITRs. In further embodiments 5’ and 3’ ITRs are present, and both ITRs are from the same serotype genome.

[0091] Recombinant adeno-associated viral vectors typically accept inserts of DNA having a size range generally about 4 kb to about 5.2 kb. If needed, stuffer sequences can be used to increase rAAV nucleic acid size and packaging efficiency. In certain embodiments the rAAV nucleic acid including stuffer is less than 5.5 kb. In further embodiments the rAAV nucleic acid including stuffer is less than 5.2 kb, less than 5.1 kb, less than 5.0 kb, less than 4.9 kb, less than 4.8 kb, less than 4.7 kb, less than 4.6 kb; between 4 kb to 5.2 kb, 3.0 kb to 5.5 kb, 4.0 kb to 5.0 kb, or 4.3 kb to 4.8 kb; or about 4.2 kb, about 4.3 kb, about 4.4 kb about, 4.5 kb, about 4.6 kb,17 4930-2481-6953.1about 4.7 kb, about 4.8 kb, about 4.9 kb, or about 5.0 kb. Preferred stuffer sequences avoid coding sequences, repetitive sequences, recombination sequences, and immunogenic sequences.

[0092] In certain embodiments the rAAV is a self-complementary adeno-associated virus vector (scAAV) or short hairpin adeno-associated virus vector (shAAV). scAAV and shAAV provide for a double-stranded rAAV nucleic acid that can be incorporated into an AAV capsid. scAAV and shAAV comprise inverted dimeric repeats providing intramolecular double-stranded DNA. scAAV can be produced by mutating an ITR terminal resolution site so that rep fails to nick the terminal resolution site. shAAV can utilize a short hairpin to produce double-stranded AAV nucleic acid. scAAV and shAAV being double-stranded DNA provide an advantage in circumventing the DNA synthesis step required for single-stranded rAAV nucleic acid upon entry into a cell. A potential disadvantage of scAAV and shAAV is the size of DNA inserts that can be incorporated is reduced by about half compared to single-stranded rAAV nucleic acid. (U.S. Patent No.10,457,940; Xie et al., Mol Ther. (2017) 25(6):1363-1374; and McCarty Mol. Ther. (2008) 16(10):1648-1656; each of which are hereby incorporated by reference herein in their entirety.)

[0093] Naturally occurring AAV capsids contain viral proteins VP1, VP2 and VP3 in a ratio of about 1:1:10. AAV vectors can be produced where all three viral proteins are based upon a particular serotypes or where one, two or all three viral protein are based on different serotypes.

[0094] Recombinant AAV capsid and nucleic acid can be based on the same serotype (or subgroup or variant), or can be different from each other. In certain embodiments, a rAAV nucleic acid has the same serotype genome (e.g., ITRs) as the encapsidating capsid protein.

[0095] In different embodiments, the rAAV capsid comprises a protein having a sequence identity of at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.9% or 100% identical to a VP1, VP2 or VP3 of any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, or AAV1 / rh.10; or VP1 of SEQ ID NO: 11 or SEQ ID NO: 14.

[0096] Recombinant AAV capsids comprising VP1 of SEQ ID NO: 11 is described, for example, in U.S. Patent No.9,8407,19; and rAAV capsids comprising VP1 of SEQ ID NO: 14 is described, for example, in U.S. Patent No.9,169,299; both of which are incorporated herein by reference.

[0097] In certain embodiments, AAV capsids comprises VP1, VP2 and VP3 each independently having a sequence identity of at least 80%, at least 90%, at least 95% or 100% to a VP1, VP2 or18 4930-2481-6953.1VP3 of any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, AAV1 / rh.10, SEQ ID NO: 11 or SEQ ID NO: 14; as well as variants (e.g., capsid variants, such as amino acid insertions, additions, substitutions and deletions) thereof. (See, for example, U.S. Patent Nos.9,909,142 and 9,840,719 disclosing RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4 and RHM15-6; U.S. Patent Publication No.2013 / 0059732 and U.S. Patent No.9,169,299, disclosing LK01, LK02, and LK03; and U.S. Patent No.11,110,153; the disclosures of which are herein incorporated in their entirety.)

[0098] In certain embodiments, the AAV capsid comprises a VP1 of SEQ ID NO: 11, a VP2 of SEQ ID NO: 12 and a VP3 of SEQ ID NO: 13.

[0099] In certain embodiments, the AAV capsid can cross the blood brain barrier and provide for CNS expression. Examples of such AAV capsids and the design of AAV capsids able to provide for CNS expression are provided in Chen et al., (2021) J. Control. Release 333, 129-138 (e.g., AAV9, AAV-PHP⋅B, AAV-PHP.eB, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV- AS, and AAV1 / rh.10), U.S. Patent No.9,585,971, and Goertsen et al., (2022) Nat. Neurosci.25, 106–115,each of which are incorporated by reference herein in its entirety.

[0100] The AAV genome contains two main genes: rep and cap. Transcription from the rep gene is initiated from two different promoters resulting in the production of nonstructural proteins designated Rep78, Rep68, Rep52, and Rep40. The rep proteins function in genome replication and / or encapsidation. The cap gene encodes for structural proteins making up the capsid (VP1, VP2 and VP3); a non-structural assembly-activating protein (APP), which performs functions related to capsid assembly; and the membrane-associated accessory protein, which may be associated with production phases of the replication cycle. (Maurer and Weitzman (2020) Hum. Gene Ther.31(9-10):499-511.)

[0101] AAV requires helper virus functions to complete its replication cycle. Helper virus functions can be supplied by different viruses in permissive cell lines. Permissive cell lines are cell lines able to support viral replication. Examples of helper viruses for AAV include adenovirus, HSV-1, HPV-16, and HBoV1, which can be used in conjunction with, for example, permissive primate cells; and baculovirus which can be used in conjunction with, for example, permissive insect cells such as sf9. (Maurer and Weitzman (2020) Hum. Gene Ther.31(9- 10):499-511 and Meier et al., (2020) Viruses 19;12(6):662.)

[0102] Recombinant AAV can be produced by supplying viral proteins needed for vector production in trans using, for example, appropriate helper viruses or plasmids and cell lines. In certain embodiments, rAAV is produced using a rAAV vector genome plasmid. The plasmid comprises that portion of the rAAV nucleic acid ultimately packaged or encapsidated to form a19 4930-2481-6953.1viral (e.g., rAAV) vector. The “plasmid backbone,” contains elements important for propagation and recombinant virus production. Except for possible 3’ ITR and / or 5’ ITR cloning remnants the plasmid backbone is not itself packaged or encapsidated into virus particles.

[0103] The vector genome plasmid may contain regions such an origin of replication and a selectable marker. Additional sites that may be present include cloning sites.

[0104] Recombinant AAV vectors can be produced from different types of cell lines including HeLa, A549, BHK, Vero, and HEK293, or derivatives thereof. In certain embodiments, HEK293 cells are used (American Type Culture Collection Accession Number ATCC CRL1573). Other host cell lines appropriate for rAAV vector production are described in, for example, Robert et al., Biotechnol. J. (2017) 12(3), 1600193; and International Application No. PCT / US2017 / 024951; the disclosures of which are herein incorporated in their entirety.

[0105] Recombinant AAV vectors can be cultured under a variety of different conditions suitable for providing cell growth and gene expression. References describing rAAV vector manufacturing include Clément and Grieger (2016) Mol. Ther. Methods Clin. Dev.16;3:16002; Robert et al., (2017) Biotechnol. J.12(3), 1600193; and Adeno-Associated Virus Vectors (2019), Ed. Castle., 1stEdition, Springer New York, NY.; each of which are hereby incorporated by reference herein in their entirety.)

[0106] In certain embodiments, AAV helper functions are introduced into the host cell by transfecting the host cell with an AAV helper construct either prior to, or concurrently with, the transfection of an AAV expression vector. A host cell having AAV helper functions can be referred to as a “helper cell” or “packaging helper cell.” AAV helper constructs are thus sometimes used to provide at least transient expression of AAV rep and / or cap genes to complement missing AAV functions necessary for productive AAV transduction. AAV helper constructs often lack AAV ITRs and can neither replicate nor package themselves. These constructs can be, for example, in the form of a plasmid, phage, transposon, cosmid, virus, or virion. A number of AAV helper constructs have been described, such as the plasmids pAAV / Ad and pIM29+45 which encode both rep and cap expression products. A number of other vectors are known which encode rep and / or cap expression products. Recombinant AAV can be produced, for example, as described in U.S. Patent 9,408,904; and International Application Nos. PCT / US2017 / 025396 and PCT / US2016 / 064414, the disclosures of which are herein incorporated in their entirety.

[0107] In certain embodiments, a rAAV vector is produced by a rAAV production cell comprising rAAV helper virus activity. The genome of the rAAV production cell comprises rAAV nucleic acid, the rep gene and the cap gene.20 4930-2481-6953.1

[0108] In certain embodiments, a rAAV vector is produced by culturing a rAAV permissive cell comprising an AAV genome plasmid, where the rAAV permissive cell comprises rep and cap genes provided either as part of the cell genome and / or by one or more separate plasmids; and helper virus activity either as part of the cell genome and / or provided by one or more separate plasmids. In further embodiments, (a) the rAAV permissive cell line is a packaging cell, wherein the genome of the packaging cell comprises the cap gene and the rep gene; (b) the rep gene, cap gene, and helper activity are provided from the same plasmid; or (c) the rep gene and cap gene are provided by a rep / cap plasmid and helper activity is provided by a helper plasmid.

[0109] In certain embodiments involving the use of HSV helper functions, the helper functions are provided by genes encoding at least UL5, UL8, UL52, and ICP8.

[0110] In certain embodiments involving the use of adenovirus helper functions, the helper functions are provided by genes encoding at least E1A, E1B19K, E1B55K, E2A, E4orf6 and VA RNA. In certain embodiments E1, E2A and VR RNA functions are provided by a helper plasmid, where additional helper functions are provided by a host strain.

[0111] In certain embodiments, rAAV vector is obtained by producing rAAV using methods described herein and purifying the rAAV vector. Purification of rAAV vector can be performed using techniques such as gradient-based purification, column-based, and combined methods. (See, e.g., Ayuso et al., (2010), Curr Gene Ther. (2010) 10(6):423-36.) V.C. Retrovirus Vectors

[0112] Retroviruses are enveloped, single-stranded RNA viruses comprising 5’ and 3’ LTRs, and a signal packaging sequence located just outside of the LTR. Different types of retrovirus vectors can contain different amounts of viral genome. In certain embodiments, the retrovirus vector is a lentiviral vector based on HIV, retaining all cis-acting sequences needed for viral RNA packaging, reverse transcription and proviral DNA integration, while removing all HIV protein-coding genes. Lentiviral vectors have a packaging capacity of up to about 9 kb. If needed, stuffer sequence can be used to increase rAAV nucleic acid size and packaging efficiency. Lentiviral vectors can be produced by supplying viral proteins needed for vector production in trans using appropriate plasmids and cell lines. (Bulcha et al., (2021) Sig. Transduct. Target Ther.6:53.) VI. Pharmaceutical Compositions

[0113] Pharmaceutical compositions comprise a pharmaceutical acceptable carrier facilitating administration and / or storage of an active agent such as an immunoglobulin G-degrading enzyme or gene therapy therapeutic. Reference to “pharmaceutically acceptable” indicates the components do not cause substantial undesirable biological effects at the amount utilized.21 4930-2481-6953.1Pharmaceutically acceptable carriers can contain different components such as one or more pharmaceutically acceptable excipients. Examples of pharmaceutically acceptable excipients include salt, sugar, buffer, solvent, preservative, protein and surfactant. A particular excipient can have more than one function. Examples of pharmaceutically acceptable excipients and carriers that can be used with viral vectors are provided in, for example, International Patent Publication Nos. WO2021 / 071835 and WO / 2024 / 138129. Examples of pharmaceutically acceptable excipients for protein are provided in, for example, Rao et al., Biological Drug Products. Pharm Res.2020 Sep 24;37(10):200. Erratum in: Pharm Res.2022 Apr;39(4):825.

[0114] Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery. Compositions suitable for parenteral administration include aqueous and non-aqueous solutions, suspensions or emulsions, which preparations are typically sterile and can be isotonic with the blood of the intended recipient. Illustrative examples include water, buffered saline, Hanks’ solution, Ringer’s solution, dextrose, fructose, ethanol, animal vegetable and synthetic oils. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.

[0115] In an embodiment, the pharmaceutical composition contains a formulation capable of injection into a subject. Examples of injectable formulation components include isotonic, sterile, saline solutions, salts (e.g., monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and mixtures of such salts), buffered saline, sugars (e.g., dextrose), and water for injection. Pharmaceutical compositions include dry, for example, freeze-dried compositions which upon addition of sterilized water or physiological saline, permit the constitution of solutions suitable for administration.

[0116] Additionally, suspensions can be prepared as appropriate oil injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension can also contain suitable stabilizers or agents which increase compound solubility facilitating the preparation of concentrated solutions.

[0117] An “effective amount” or “sufficient amount” refers to an amount providing an indicated or desired effect. The effective amount can be administered in single or multiple doses, alone or in combination, with one or more other compositions (e.g., additional therapeutic or immunosuppressive agents), treatments, protocols, or therapeutic regimens; and provide for a long or short term response.

[0118] Pharmaceutical compositions comprising transgene-encoding polynucleotide can be delivered to a subject, to allow production of the encoded protein. In certain embodiments,22 4930-2481-6953.1pharmaceutical compositions comprise sufficient genetic material to enable a recipient to produce a therapeutically effective amount of a therapeutic agent in the subject.

[0119] A “therapeutically effective amount” refers to an amount of an active ingredient or component that elicits the desired or indicated biological or medicinal response in a subject. In certain embodiments, a therapeutically effective amount decreases observed symptoms and / or biomarkers associated with a particular disease or disorder. Selection of a particular effective dose can be enhanced taking into account different factors, including the disease or disorder to be treated or prevented, the symptoms involved, safety and effectiveness in animal models, the patient’s body mass, and the patient’s immune status. The optimal dose to be employed will also depend on the route of administration, and the severity of the disease or disorder, and can be evaluated depending upon patient’s circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0120] Pharmaceutical compositions comprising an immunoglobulin G-degrading enzyme can be delivered to reduce the IgG protein in the subject. In different embodiments, the immunoglobulin G-degrading enzyme pharmaceutical composition is provided to assist in gene therapy, transplantation, or treating an autoimmune disease.

[0121] Additional guidance and examples of pharmaceutical compositions and delivery systems are provided in, for example, Remington: The Science and Practice of Pharmacy (2020) 23th ed., University of the Sciences in Philadelphia, published by Elsevier; The Merck Index (2013) 15th ed., Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technomic Publishing Co., Inc., Lancaster, Pa.; and Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11th ed., Lippincott Williams & Wilkins, Baltimore, MD. VII. Administration and Treatment

[0122] Immunoglobulin G-degrading enzymes can be used, for example, as a therapeutic for the treatment of diseases or disorders mediated by an IgG immune response, to facilitate treatment that may evoke IgG immune response, and for research purposes. Treatments expected to produce an IgG response include transplantation and the use of gene delivery vehicles, such as viral vectors, that can evoke an immune response.

[0123] In certain embodiments an immunoglobulin G-degrading enzyme is administered as a therapeutic for the treatment of a disease or disorder mediated by an IgG immune response. In different embodiments the disease or disorder is selected from the group consisting of: Addison's disease; anti-GBM glomerulonephritis (related to Goodpasteur); anti-neutrophil cytoplasmic antibody-associated vasculitides (ANCA associated vasculitis); Wegener granulomatosis; Churg-Strauss syndrome; microscopic polyangiitis; anti-NMDAR Encephalitis; anti-23 4930-2481-6953.1phospholipid antibody syndrome (APS) and catastrophic APS; autoimmune bullous skin diseases; Pemphigus; Pemphigus foliaceus (PF); fogo selvagem (FS) (endemic form); pemphigus vulgaris (PV); autoimmune hemolytic anemia (AIHA); autoimmune hepatitis (AIH); autoimmune neutropenia (AIN); bullous pemphigoid (BP); Celiac disease; chronic utricaria; complete congenital heart block (CCHB); Epidermolysis bullosa acquisita (EBA); essential mixed cryoglobulinemia; Goodpasture 's syndrome (also known as Goodpasture 's disease and anti-glomerular basement membrane disease); Graves' disease (Basedow's disease), Goitre and hyperthyroidism, infiltrative exopthalmos and infiltarative dermopathy; Guillain-Barre syndrome (GBS); acute inflammatory demyelinating polyneuropathy (AIDP); acute motor axonal neuropathy (AMAN); hemophilia - acquired FVIII deficiency; idiopathic thrombocytopenic purpura (ITP); Lambert-Eaton myasthenic syndrome (LEMS); Mixed Connective Tissue Disease (MCTD); Multiple Myeloma; Myasthenia gravis; Myasthenic crisis; Myocarditis, dilated cardiomyopathy (DCM) (congestive cardiomyopathy); Neuromyelitis Optica (NMO); primary biliary cirrhosis (PBC); Primary Progressive Multiple Sclerosis (PPMS); rheumatic heart disease (RHD); rheumatic fever; rheumatoid arthritis (RA); serum- sickness, immune complex hypersensitivity (type III); Sjogren Syndrome (SS); SLE including Lupus nephritis; Stiff-person syndrome (SPS); Systemic sclerosis (scleroderma); transplant rejection; and thrombotic thrombocytopenic purpura (TIP). (See U.S. Patent No.10,696,959 hereby incorporated by reference herein in its entirety.)

[0124] In certain embodiments an immunoglobulin G-degrading enzyme is administered to reduce an immune response to organ or tissue transplantation, for example, as part of a treatment involving organ or tissue transplantation. In a further embodiment, the enzyme is used in conjunction with liver transplantation.

[0125] In certain embodiments, the immunoglobulin G-degrading enzyme is administered in conjunction with gene therapy or other treatment that can evoke an immune response. Gene therapy treatments and targets are illustrated below and / or in Section IV supra (including IV.A, IV.B and IV.C. supra). In further embodiments, gene therapy involves the use of a viral vector, such those illustrated in Section V supra (including V.A., V.B., and V.C.).

[0126] Reference to “treatment” or “treat” refers to both prophylactic treatment, and therapeutic treatment of a patient having a disease or disorder. Reference to “prophylactic” treatment indicates a decrease in the likelihood of contracting a disease or disorder or decreasing the potential severity of a disease or disorder. Reference to “therapeutic” indicates a clinical meaningful amelioration in at least one symptom or cause associated with a disease or disorder. Thus, treatments include administration to subjects at risk of contracting the disease or disorder,24 4930-2481-6953.1suspected to have contracted the disease or disorder, as well as subjects who are ill or have been diagnosed as suffering from a disease or disorder and includes suppression of clinical relapse.

[0127] The terms “ameliorate”, and “amelioration” refer to a detectable or measurable improvement in a disease or disorder symptom or an underlying cellular response. A detectable or measurable improvement includes a subjective or objective decrease, reduction, inhibition, suppression, limit or control in the occurrence, frequency, severity, progression, or duration of the disease or disorder, or complication caused by or associated with the disease or disorder, or an improvement in a symptom or an underlying cause or a consequence of the disease or disorder, or a reversal of the disease or disorder.

[0128] An effective amount of an immunoglobulin G-degrading enzyme, is an amount sufficient to degrade native IgG in a subject. When used as a therapeutic, in different embodiments, the amount is sufficient to cause at least a 10%, at least 20%, or at least 30% reduction in IgG. When used to a facilitate treatment that may evoke an IgG immune response, in different embodiments, the amount is sufficient to prevent an increase in IgG by more than 25%, or cause a decrease in IgG of at least 25%.

[0129] Preferably, an “effective amount” or “sufficient amount” of an agent having a direct therapeutic effect, is an amount sufficient to result in a clinical meaningful amelioration in at least one symptom or cause associated with a disease or disorder.

[0130] An effective amount can be administered alone or in combination with another composition, treatment, protocol, or therapeutic regimen. The amount can be proportionally increased, for example, based on the need of the subject, type, status and severity of the disease or disorder treated or side effects.

[0131] An effective amount or a sufficient amount need not be effective in each and every subject treated, nor a majority of treated subjects in a given group or population. An effective amount or a sufficient amount means effectiveness or sufficiency in a particular subject, not a group or the general population. As is typical for such methods, some subjects may exhibit a greater response, or less or no response to a given treatment method or use.

[0132] In certain embodiments, the immunoglobulin G-degrading enzyme is administered at a dosage of about 0.01 mg / kg to about 10 mg / kg body weight of a subject. For example, in different embodiments a suitable dosage may be from about 0.05 mg / kg to about 5 mg / kg body weight of a subject, or from about 0.1 mg / kg to about 4 mg / kg body weight of a subject.

[0133] Preferred treatment doses for gene therapy can vary depending on the particular vector and different factors such as the type, onset, progression, severity, frequency, duration or particular disease or disorder to which treatment is directed; the clinical endpoint desired; previous or simultaneous treatments; and the general health, age, gender, race or immunological25 4930-2481-6953.1competency of the subject. The preferred dose to achieve a therapeutic effect, will also vary based on several factors including route of administration, the level of transgene expression required to achieve a therapeutic effect, the host immune response to the gene delivery vehicle, host immune response transgene expression product, and the stability of the protein, peptide, or nucleic acid expressed. The dose amount, number, frequency, or duration can be proportionally increased or reduced, as indicated by adverse side effects, complications or other risk factors of the treatment or therapy and the status of the subject.

[0134] The overall level of transgene expression can vary depending upon the employed vector. In different embodiments of gene therapy providing a therapeutic protein, the provided expression or activity is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% of normal expression of the corresponding subject protein.

[0135] In certain embodiments, expression or activity of a targeted protein is reduced. In different embodiments reduction of expression or activity of a protein targeted by a therapeutic nucleic acid is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of normal expression of the target protein.

[0136] In different embodiments a suitable viral vector dosage is from about 0.01 mg / kg to about 10 mg / kg of vector per kg body weight of a subject, about 0.01 mg / kg to about 0.1 mg / kg of vector per kg body weight of a subject, about 0.1 mg / kg to about 1.0 mg / kg of vector per kg body weight of a subject, or about 1.0 mg / kg to about 10 mg / kg of vector per body weight of a subject.

[0137] Generally, rAAV vector doses range from at least 1x108vector genomes per kilogram (vg / kg) of the weight of the subject, or more, for example, 1x109, 1x1010, 1x1011, 1x1012, 1x1013or 1x1014, or more, vector genomes per kilogram (vg / kg) of the weight of the subject, to achieve a therapeutic effect. In different embodiments the rAAV dose is about 5x1011rAAV vg / kg or greater than about 5x1011rAAV vg / kg; about 1x1012rAAV vg / kg or greater than about 1x1012rAAV vg / kg; about 2x1012rAAV vg / kg or greater than about 2x1012rAAV vg / kg; about 3x1012rAAV vg / kg or greater than about 3x1012rAAV vg / kg; about 4x1012rAAV vg / kg or greater than about 4x1012rAAV vg / kg; about 5x1012rAAV vg / kg or greater than about 5x1012rAAV vg / kg; about 1x1013rAAV vg / kg or greater than about 1x1013rAAV vg / kg; about 2x1013rAAV vg / kg or greater than about 2x1013rAAV vg / kg; about 3x1013rAAV vg / kg or greater than about 3x1013rAAV vg / kg; about 4x1013rAAV vg / kg or greater than about 4x1013rAAV vg / kg; about26 4930-2481-6953.15x1013rAAV vg / kg or greater than about 5x1013rAAV vg / kg; or about 6x1013rAAV vg / kg or greater than about 6x1013rAAV vg / kg.

[0138] Examples of dose ranges of rAAV vg / kg include a dose range from about 5x1011to about 6x1013rAAV vg / kg; a dose range from about 5x1011to about 5.5x1011rAAV vg / kg; a dose range from about 5.5x1011to about 6x1011rAAV vg / kg; a dose range from about 6x1011to about 6.5x1011rAAV vg / kg; a dose range from about 6.5x1011to about 7x1011rAAV vg / kg; a dose range from about 7x1011to about 7.5x1011rAAV vg / kg; a dose range from about 7.5x1011to about 8x1011rAAV vg / kg; a dose range from about 8x1011to about 8.5x1011rAAV vg / kg; a dose range from about 8.5x1011to about 9x1011rAAV vg / kg; a dose range from about 9x1011to about 9.5x1011rAAV vg / kg; a dose range from about 9.5x1011to about 1x1012rAAV vg / kg; a dose range from about 1x1012to about 1.5x1012rAAV vg / kg; a dose range from about 1.5x1012to about 2x1012rAAV vg / kg; a dose range from about 2x1012to about 2.5x1012rAAV vg / kg; a dose range from about 2.5x1012to about 3x1012rAAV vg / kg; a dose range from about 3x1012to about 3.5x1012rAAV vg / kg; a dose range from about 3.5x1012to about 4x1012rAAV vg / kg; a dose range from about 4x1012to about 4.5x1012rAAV vg / kg; a dose range from about 4.5x1012to about 5x1012rAAV vg / kg; a dose range from about 5x1012to about 5.5x1012rAAV vg / kg; a dose range from about 5.5x1012to about 6x1012rAAV vg / kg; a dose range from about 6x1012to about 6.5x1012rAAV vg / kg; a dose range from about 6.5x1012to about 7x1012rAAV vg / kg; a dose range from about 7x1012to about 7.5x1012rAAV vg / kg; a dose range from about 7.5x1012to about 8x1012rAAV vg / kg; a dose range from about 8x1012to about 8.5x1012rAAV vg / kg; a dose range from about 8.5x1012to about 9x1012rAAV vg / kg; a dose range from about 9x1012to about 9.5x1012rAAV vg / kg; a dose range from about 9.5x1012to about 1x1013rAAV vg / kg; a dose range from about 1x1013to about 1.5x1013rAAV vg / kg; a dose range from about 1.5x1013to about 2x1013rAAV vg / kg; a dose range from about 2x1013to about 2.5x1013rAAV vg / kg; a dose range from about 2.5x1013to about 3x1013rAAV vg / kg; a dose range from about 3x1013to about 3.5x1013rAAV vg / kg; a dose range from about 3.5x1013to about 4x1013rAAV vg / kg; a dose range from about 4x1013to about 4.5x1013rAAV vg / kg; a dose range from about 4.5x1013to about 5x1013rAAV vg / kg; a dose range from about 5x1013to about 5.5x1013rAAV vg / kg; a dose range from about 5.5x1013to about 6x1013rAAV vg / kg; or a dose range from about 6x1013to about 1x1014rAAV vg / kg.

[0139] In certain embodiments, rAAV vg / kg are administered at a dose of about 5x1011vg / kg, about 6x1011vg / kg, about 7x1011vg / kg, about 8x1011vg / kg, about 9x1011vg / kg, about 1x1012vg / kg, about 2x1012vg / kg, about 3x1012vg / kg, about 4x1012vg / kg, about 5x1012vg / kg, about 6x1012vg / kg, about 7x1012vg / kg, about 8x1012vg / kg, about 9x1012vg / kg, about 1x1013vg / kg,27 4930-2481-6953.1about 2x1013vg / kg, about 3x1013vg / kg, about 4x1013vg / kg, about 5x1013vg / kg, or about 6x1013vg / kg.

[0140] Delivery and administration can be systemically, regionally or locally, for example, by injection or infusion. Delivery of the compositions in vivo can generally be accomplished, for example, by injection using a conventional syringe, although other delivery methods such as convection-enhanced delivery are envisioned (see, e.g., U.S. Patent No.5,720,720). Depending on the composition delivery, for example, can be subcutaneously, epidermally, intradermally, intrathecally, intraorbitally, intramucosally, intraperitoneally (IP), intravenously (IV), intra- pleurally, intraarterially, orally, intrahepatically, via the portal vein, intracranial or intramuscularly.

[0141] In certain embodiments, the immunoglobulin G-degrading enzyme is used in conjugation with gene therapy treating a lung disease (e.g., cystic fibrosis), a blood disorder (e.g., anemia), CNS diseases and disorder, epilepsy, a lysosomal storage disease (e.g., aspartylglucosaminuria), Batten disease, late infantile neuronal ceroid lipofuscinosis type 2 (CLN2), cystinosis, Fabry disease, Gaucher disease types I, II, and III, glycogen storage disease II (Pompe disease), GM2- gangliosidosis type I (Tay-Sachs disease), GM2-gangliosidosis type II (Sandhoff disease), mucolipidosis types I (sialidosis type I and II), II (I-cell disease), III (pseudo-Hurler disease) and IV, mucopolysaccharide storage diseases (Hurler disease and variants, Hunter, Sanfilippo Types A, B, C, D, Morquio Types A and B, Maroteaux-Lamy and Sly diseases), Niemann-Pick disease types A / B, C1 and C2, and Schindler disease types I and II), hereditary angioedema (HAE), a copper or iron accumulation disorder (e.g., Wilson’s or Menkes disease), lysosomal acid lipase deficiency, a neurological or neurodegenerative disorder, cancer, type 1 or type 2 diabetes, adenosine deaminase deficiency, a metabolic defect (e.g., glycogen storage diseases), and a disease of solid organs (e.g., brain, liver, kidney, or heart).

[0142] In certain embodiments, the immunoglobulin G-degrading enzyme is used in conjugation with gene therapy treating glycogen storage disease type II (Pompe disease). Pompe disease is an autosomal recessive disorder caused by mutations in the gene encoding the lysosomal enzyme acid α-glucosidase (GAA), which catalyzes the degradation of glycogen. The resulting enzyme deficiency leads to pathological accumulation of glycogen and lysosomal alterations in body tissues, resulting in cardiac, respiratory, and skeletal muscle dysfunction.

[0143] In further embodiments for Pompe disease, an effective amount is an amount of GAA that inhibits or reduces glycogen production or accumulation, enhances or increases glycogen degradation or removal, reduces lysosomal alterations in tissues of the body of a subject, or improves muscle tone and / or muscle strength and / or respiratory function in a subject. Effective amounts can be determined, for example, by ascertaining the kinetics of GAA uptake by28 4930-2481-6953.1myoblasts from plasma. Myoblasts GAA uptake rates (K uptake) of about 141 – 147 nM appear to be effective (e.g., Maga et al., J. Biol. Chem.2012, 8;288(3), 1428). In animal models, GAA activity levels in plasma greater than about 1,000 nmol / hr / mL, for example, about 1,000 to about 2,000 nmol / hr / mL have been observed to be therapeutically effective.

[0144] In certain embodiments, the immunoglobulin G-degrading enzyme is used in conjugation with gene therapy treating blood clotting disorders such as hemophilia A, hemophilia A with inhibitory antibodies, hemophilia B, hemophilia B with inhibitory antibodies, a deficiency in any coagulation Factor: VII, VIII, IX, X, XI, V, XII, II, von Willebrand factor, or a combined FV / FVIII deficiency, thalassemia, vitamin K epoxide reductase C1 deficiency or gamma- carboxylase deficiency.

[0145] In further embodiments directed to hemophilia A and hemophilia B treatment, blood coagulation factor concentration greater than 1% of factor concentration found in a normal individual is needed to change a severe disease phenotype to a moderate one. A severe phenotype is characterized by joint damage and life-threatening bleeds. To convert a moderate disease phenotype into a mild one, it is expected that a blood coagulation factor concentration greater than 5% of normal is needed.

[0146] In further embodiments directed to FVIII and FIX, a therapeutic effect is achieved by providing the total amount of FVIII or FIX in the subject / human to be greater than 1% of the FVIII or FIX present in normal subjects / humans, e.g., 1% of 100-300 ng / mL. FVIII normal level is about 100-200 ng / ml and FIX levels in normal humans is 5000 ng / ml, but levels can be more or less and still be considered normal. Functional clotting can be determined, for example, by an activated partial thromboplastin time (aPTT) one-stage clotting assay.

[0147] In certain embodiments, the immunoglobulin G-degrading enzyme is used in conjugation with gene therapy to treat bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC); over- anticoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, small molecule antithrombotics (i.e., FXa inhibitors), or a platelet disorder such as, Bernard Soulier syndrome, Glanzmann thrombasthenia, or storage pool deficiency.

[0148] In certain embodiments, the immunoglobulin G-degrading enzyme is used in conjugation with gene therapy to treat a proliferative disease (e.g., cancers, tumors and dysplasias), Crigler- Najjar and metabolic diseases like metabolic diseases of the liver; Friedreich ataxia; infectious diseases; viral diseases induced for example by hepatitis B or C viruses, HIV, herpes, and retroviruses; genetic diseases such as cystic fibrosis, dystroglycanopathies, myopathies such as Duchenne muscular myopathy or dystrophy, myotubular myopathy, sickle-cell anemia, sickle cell disease, Fanconi’s anemia, diabetes, amyotrophic lateral sclerosis (ALS), myotubularin29 4930-2481-6953.1myopathy, motor neuron diseases such as spinal muscular atrophy (SMA), spinobulbar muscular atrophy, or Charcot-Marie-Tooth disease; arthritis; severe combined immunodeficiencies such as RS-SCID, ADA-SCID or X-SCID; Wiskott-Aldrich syndrome; X-linked thrombocytopenia; X- linked congenital neutropenia; chronic granulomatous disease; clotting factor deficiencies; cardiovascular disease such as restenosis, ischemia, dyslipidemia, and homozygous familial hypercholesterolemia; eye or ocular diseases such as retinitis pigmentosa, X-linked retinitis pigmentosa, autosomal dominant retinitis pigmentosa, recessive retinitis pigmentosa, choroideremia, choroidal neovascularization, gyrate atrophy, retinoschisis, X-linked retinoschisis, macular degeneration, diabetic macular edema (DME), diabetic retinopathy associated with DME, wet age-related macular degeneration (wet AMD or wAMD), macular edema following retinal vein occlusion, non-arteritic ischaemic optic neuropathy, Leber congenital amaurosis, Leber hereditary optic neuropathy, achromatopsia, and Stargardt disease; lysosomal storage diseases such as San Filippo syndrome; hyperbilirubinemia such as CN type I or II or Gilbert’s syndrome; glycogen storage disease such as GSDI, GSDIII, GSDIV, GSDV, GSDVI, GSDVII, GSDVIII or lethal congenital glycogen storage disease of the heart.

[0149] In certain embodiments, the immunoglobulin G-degrading enzyme is used in conjugation with gene therapy that affects or originates in the central nervous system (CNS). In certain embodiments, the disease is a neurodegenerative disease. Non-limiting examples of CNS or neurodegenerative disease include Alzheimer’s disease, Huntington’s disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy’s disease, a polyglutamine repeat disease, and Parkinson’s disease. In certain embodiments, the disease is a psychiatric disease, an addiction (e.g., to tobacco, alcohol, or drug), epilepsy, Canavan’s disease, or adrenoleukodystrophy. In certain embodiments, the CNS or neurodegenerative disease is a polyglutamine repeat disease such as spinocerebellar ataxia (SCA1, SCA2, SCA3, SCA6, SCA7, or SCA17).

[0150] In certain embodiments the immunoglobulin G-degrading enzyme is administered at about the same time as a treatment that may evoke an immune response, such as viral vector gene therapy or transplantation; or (1) up to about 5 minutes, (2) up to about 15 minutes, (3) up to about 30 minutes, (4) up to about 45 minutes, (5) up to about 60 minutes, (6) up to about 90 minutes, (7) up to about 2 hours, (8) up to about 3 hours, (9) up to about 4 hours, (10) up to about 5 hours, (11) up to about 6 hours, (12) up to about 7 hours, (13) up to about 8 hours, (14) up to about 9 hours, (15) up to about 10 hours, (16) up to about 12 hours, (17) up to about 24 hours, (18) up to about 48 hours, (19) up to about 72 hours, (20) up to about 96 hours, or (21) up to about a week prior to the treatment. In further embodiments, the immunoglobulin G- degrading enzyme protein is administered within at least about 60%, at about least 70%, at about30 4930-2481-6953.1least 80%, at about least 90%, at about least 95% or at about the times indicated for any of (1) to (21). For examples, administration ranges for (1) up to five minutes in different embodiments include about 3-5 minutes, about 3.75 – 5 minutes, about 4-5 minutes, about 4.5-5 minutes, or about 5 minutes.

[0151] In certain embodiments the immunoglobulin G-degrading enzyme is administered at about the same time, or after a treatment that may evoke an immune response, or after an adverse immune response is observed; or (1) up to about 5 minutes, (2) up to about 15 minutes, (3) up to about 30 minutes, (4) up to about 45 minutes, (5) up to about 60 minutes, (6) up to about 90 minutes, (7) up to about 2 hours, (8) up to about 3 hours, (9) up to about 4 hours, (10) up to about 5 hours, (11) up to about 6 hours, (12) up to about 7 hours, (13) up to about 8 hours, (14) up to about 9 hours, (15) up to about 10 hours, (16) up to about 12 hours, (17) up to about 24 hours, (18) up to about 48 hours, (19) up to about 72 hours, (20) up to about 96 hours, (21) up to about a week, or (22) greater than a week. In further embodiments, the immunoglobulin G- degrading enzyme protein is administered within at least about 60%, at about least 70%, at about least 80%, at about least 90%, at about least 95% or at about the times indicated in any of (1) to (22). VIII. Kits

[0152] Further provided herein is a kit comprising in separate containers: (a) a pharmaceutical composition comprising an immunoglobulin G-degrading enzyme produced using a codon optimized encoding sequence; and (b) a gene delivery vehicle comprising a therapeutic transgene. In certain embodiments both containers are in the same location and may also be provided with a label with instructions for administration according to the methods described herein IX. Additional Aspects and Embodiments

[0153] Additional aspects, embodiments, and examples of combinations thereof include the following:

[0154] A first aspect describes a polynucleotide comprising a nucleic acid sequence encoding a protein comprising the sequence of SEQ ID NO: 9 or a variant of SEQ ID NO: 9 having 1, 2, 3, 4 or 5 N-terminal amino acids replaced with a signal peptide (“signal peptide variant thereof”) wherein: (a) each codon encoding for asparagine in the protein is AAC; and (b) each codon encoding for serine in the protein is TCT.31 4930-2481-6953.1

[0155] A first Embodiment (E1) further describes the first aspect wherein the protein consists of the amino acid sequence of SEQ ID NO: 9.

[0156] A second Embodiment (E2) further describes the first aspect and E1 wherein each codon encoding for proline in said protein is CCG.

[0157] A third Embodiment (E3) further describes the first aspect, E1 and E2, wherein each codon encoding for threonine is ACT.

[0158] A fourth Embodiment (E4) further describes the first aspect, E1, E2, and E3 wherein each codon encoding for histidine is CAC.

[0159] A second aspect describes a polynucleotide comprising a nucleic acid sequence encoding a protein sequence comprising the sequence of SEQ ID NO: 9, wherein the nucleic acid sequence encoding the protein comprises in positions corresponding to SEQ ID NO: 1: a) codons 3, 5, 13, 22, 122, 137, 159, 172, 190, 195, 208, 224, 245, 257, 259, 262, 274,81, 301, and 306 encoding for serine are each TCT; and b) codons 7, 33, 48, 59, 73, 82, 100, 102, 117, 154, 162, 203, 229, 233, 237, 246, 248, 260, 273, 289, 308, and 311 encoding for asparagine are each AAC.

[0160] Codon numbering is provided in FIGs.1A, 1B and 1C, and provides the corresponding location.

[0161] A fifth Embodiment (E5) further describes the second aspect, wherein the nucleic acid sequence encoding said protein comprises in positions corresponding to SEQ ID NO: 1: codons 17, 30, 31, 44, 94, 134, 145, 165, 167, and 175, encoding for proline are each CCG.

[0162] A sixth Embodiment (E6) further describes the second aspect and E5, wherein the nucleic acid sequence encoding said protein comprises in positions corresponding to SEQ ID NO: 1: codons 16, 21, 25, 29, 35, 55, 57, 70, 115, 138, 161, 166, 185, 194, 216, 226, 255, 299, 302, and 310 encoding for threonine are each ACT.

[0163] A seventh Embodiment (E6) further describes the second aspect, E5, and E6, wherein the nucleic acid sequence encoding said protein comprises in positions corresponding to SEQ ID NO: 1: codons 19, 42, 76, 93, 118, 140, 147, 163, 197, 225, and 234 encoding for histidine are each CAC.

[0164] An eighth Embodiment (E8) further describes the first aspect, the second aspect, E1, E2, E3, E4, E5, E6 and E7 wherein the nucleic acid sequence encoding for the protein comprises a sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, of at least 97% identical, to any of SEQ ID NOs: 1-8. Reference to a sequence identity, or another property, for different reference sequences provides that each reference sequence independently has the indicated property. In further embodiments the nucleic acid sequence encoding for the protein comprises a sequence at least 80% identical, at least 85% identical, at32 4930-2481-6953.1least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 3 or comprises SEQ ID NO3: or is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 4 or comprises SEQ ID NO: 4.

[0165] A nineth Embodiment (E9) further describes the first aspect, the second aspect, E1, E2, E3, E4, E5, E6 and E7, wherein the nucleic acid sequence encoding for the protein differs from any of SEQ ID NOs: 3-8 by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides; or comprises any of SEQ ID NOs: 3-8. As noted in E8, reference to a sequence identity, or another property, for different reference sequences provides that each reference sequence independently has the indicated property. A couple of examples, for illustration purposes, are comprising SEQ ID NO: 3 or differing from SEQ ID NO: 3 by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides; and comprising SEQ ID NO: 4 or differing from SEQ ID NO: 4 by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0166] A third aspect describes a polynucleotide comprising a nucleic acid sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 9, wherein the nucleic acid sequence is at least 90% identical to any of SEQ ID NOs: 1-8. In further embodiments, the nucleic acid sequence is at least 95% identical to any of SEQ ID NO: 1-8; at least 96% identical to any of SEQ ID NO: 1-8; at least 97% identical to any of SEQ ID NO: 1-8; at least 98% identical to any of SEQ ID NO: 1-8; at least 99% identical to any of SEQ ID NOs: 1-8; differs from any of SEQ ID NOs: 1-8 by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides; or comprises the sequence of any of SEQ ID NOs: 1-8.

[0167] A tenth embodiment (E10) further describes the first aspect, the second aspect, the third aspect E1, E2, E3, E4, E5, E6, E7, E8, and E9, wherein the nucleic acid sequence encoding the protein comprising the amino acid sequence of SEQ ID NO: 9, or signal peptide variant thereof, comprises a stop codon at the 3’ end of the sequence of SEQ ID NO: 9. DNA stop codons have the sequence of TAA, TAG, and TGA. In further embodiments, multiple stop codons are provided at the 3’ end.

[0168] An eleventh embodiment (E11) further describes the first aspect, the second aspect, the third aspect, E1, E2, E3, E4, E5, E6, E7, E8, E9 and E10, wherein the polynucleotide in an expression cassette further comprises a promoter operatively linked to the nucleic acid sequence encoding the protein comprising the sequence of SEQ ID NO: 9 or signal peptide variant thereof.

[0169] A twelfth embodiment (E12) further describes E11, wherein the expression cassette comprises a Shine-Dalgarno sequence operatively linked to the nucleic acid sequence encoding the protein comprising the sequence of SEQ ID NO: 9 or signal peptide variant thereof. In a33 4930-2481-6953.1further embodiment, the expression cassette comprises a polyadenylation sequence operatively linked to the nucleic acid sequence encoding the protein comprising the sequence of SEQ ID NO: 9 or signal peptide variant thereof.

[0170] A fourth aspect is directed to a plasmid comprising the polynucleotide of any of the first aspect, the second aspect, the third aspect, E1, E2, E3, E4, E5, E6, E7, E8, E9, E10, E11 and E12, wherein the plasmid further comprises an origin of replication and selectable marker.

[0171] A fifth aspect is directed to a bacterial artificial chromosome or bacteriophage nucleic acid comprising the polynucleotide of any of the first aspect, the second aspect, the third aspect, E1, E2, E3, E4, E5, E6, E7, E8, E9, E10, E11 and E12.

[0172] A sixth aspect is directed to a recombinant prokaryotic cell comprising the polynucleotide, expression cassette, plasmid, bacterial artificial chromosome, or bacteriophage nucleic acid of any of the first aspect, the second aspect, the third aspect, the fourth aspect, the fifth aspect, E1, E2, E3, E4, E5, E6, E7, E8, E9, E10, E11 and E12.

[0173] A seventh aspect is directed to a method of producing a protein comprising the sequence of SEQ ID NO: 9 or a deletion variant thereof lacking the N-terminal methionine, where the method comprises culturing the recombinant prokaryotic cell of the sixth aspect.

[0174] An eighth aspect is directed to obtaining a protein comprising the sequence of SEQ ID NO: 9 or a deletion variant thereof lacking the N-terminal methionine, comprising culturing the recombinant prokaryotic cell of the sixth aspect and purifying the protein.

[0175] A thirteenth embodiment (E13) further describes the eighth aspect, wherein the purity of the protein using reverse phase liquid chromatography is at least 85%, at least 90%, or at least 95%.

[0176] A fourteenth embodiment (E14) further describes the eighth aspect, wherein the purity of the protein using size exclusion chromatography is at least 94%, at least 95%, at least 96%, at least 97% or at least 98%.

[0177] A fifteenth embodiment (E15) further describes the eighth aspect, wherein the purity of the protein using CE-SEC reduced chromatography is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%.

[0178] A nineth aspect is directed to a method of preparing a pharmaceutical composition comprising a protein comprising the amino acid sequence of SEQ ID NO: 9 or a deletion variant thereof lacking the N-terminal methionine, comprising the steps: (a) producing the protein comprising the sequence of SEQ ID NO: 9 or a deletion variant thereof lacking the N-terminal methionine, according to the seventh aspect; (b) purifying the protein according to any of the eighth aspect or E13-E15; and (c) combining the purified protein with a pharmaceutically acceptable carrier.34 4930-2481-6953.1

[0179] A tenth aspect is directed to a method of administering a viral vector to a subject comprising the steps of: (a) preparing a protein pharmaceutical composition comprising a protein comprising the amino acid sequence of SEQ ID NO: 9 or a deletion variant thereof lacking the N- terminal methionine, according to nineth aspect; (b) administering the protein pharmaceutical composition prepared in step (a) to the subject; and (c) administering the viral vector to the subject; wherein step (b) can be performed prior to, after, or at the time as step (c).

[0180] A sixteenth embodiment (E16) further describes the tenth aspect, wherein viral vector is a recombinant adeno-associated viral vector comprising nucleic acid encoding a therapeutic protein or a biologically active nucleic acid.

[0181] An eleventh aspect is directed to method of treating an autoimmune disease is a subject comprising the steps of: (a) preparing a protein pharmaceutical composition comprising the amino acid sequence of SEQ ID NO: 9 or a deletion variant thereof lacking the N-terminal methionine, according to nineth aspect; and (b) administering the protein pharmaceutical composition prepared in step (a) to the subject.

[0182] A twelfth aspect is directed to a method of organ or tissue transplantation to a subject comprising the steps of: (a) preparing a protein pharmaceutical composition comprising the amino acid sequence of SEQ ID NO: 9 or a deletion variant thereof lacking the N-terminal methionine, according to the nineth aspect; (b) administering the protein pharmaceutical composition prepared in step (a) to the subject; and (c) transferring the organ or tissue to the subject, wherein step (b) can be performed prior to, after, or at the time as step (c).

[0183] A seventeenth embodiment (E17) further describes the twelfth aspect, wherein the method is used in liver transplantation.

[0184] An eighteenth embodiment (E18) further describes the tenth aspect, eleventh aspect, twelfth aspect, E16 and E17, wherein the subject is a human.35 4930-2481-6953.1X. Sequences

[0185] Table 1 includes different nucleic acid encoding the protein of SEQ ID NO: 9. In certain embodiments, a polynucleotide encodes a protein comprising the sequence of SEQ ID NO: 9 and comprises a nucleic acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of the nucleic acid sequences provided in Table 1, Some nucleic acid sequences provided in Table 1 comprise a stop codon, illustrated in bold. In certain embodiments an alternative, or multiple stop codons are used. Table 136 4930-2481-6953.137 4930-2481-6953.138 4930-2481-6953.139 4930-2481-6953.1EXAMPLES

[0186] Examples are provided below further illustrating different features of the present invention and methodology for practicing the invention. The provided examples do not limit the claimed invention.

[0187] Example 1: Protein Expression and Characterization

[0188] Plasmids containing different nucleic acid sequences encoding for IdeS of SEQ ID NO: 9 were transfected into E.coli and grown in either an AMBR® or 20L fermentation vessel and purified. End of fermentation (EoF), polyethyleneimine (PEI) filtrate product titre, and BDS were measured. The results are shown in Table 2. Table 2

[0189] IdeS protein characterization results are provided in Table 3. Table 340 4930-2481-6953.1

[0190] Based on the intact mass, the product purified from cells transfected with plasmid comprising SEQ ID NO: 2 was a contaminant.

[0191] While the invention has been described and illustrated with reference to certain particular embodiments thereof, those skilled in the art will appreciate that various adaptations, changes, modifications, substitutions, deletions, or additions of procedures and protocols may be made without departing from the spirit and scope of the invention.41 4930-2481-6953.1

Claims

CLAIMS I / we claim:

1. A polynucleotide comprising a nucleic acid sequence encoding a protein comprising the sequence of SEQ ID NO: 9 or a signal peptide variant of SEQ ID NO: 9 having 1, 2, 3, 4 or 5 N- terminal amino acids replaced with a signal peptide, wherein: (a) each codon encoding for asparagine in said protein is AAC; and (b) each codon encoding for serine in said protein is TCT.

2. The polynucleotide of claim 1, wherein said protein consists of said amino acid sequence of SEQ ID NO:

9.

3. The polynucleotide of claim 1, wherein: (c) each codon encoding for proline in said protein is CCG; (d) each codon encoding for threonine in said protein is ACT; and (e) each codon encoding for histidine in said protein is CAC.

4. The polynucleotide of claim 2, wherein: (c) each codon encoding for proline in said protein is CCG; (d) each codon encoding for threonine in said protein is ACT; and (e) each codon encoding for histidine in said protein is CAC.

5. A polynucleotide comprising a nucleic acid sequence encoding a protein comprising the sequence of SEQ ID NO: 9, wherein said nucleic acid sequence encoding said protein comprises in positions corresponding to SEQ ID NO: 1: (a) codons 3, 5, 13, 22, 122, 137, 159, 172, 190, 195, 208, 224, 245, 257, 259, 262, 274, 281, 301, and 306 encoding for serine are each TCT; and (b) codons 7, 33, 48, 59, 73, 82, 100, 102, 117, 154, 162, 203, 229, 233, 237, 246, 248, 260, 273, 289, 308, and 311 encoding for asparagine are each AAC.

6. The polynucleotide of claim 5, wherein said nucleic acid sequence encoding said protein comprises in positions corresponding to SEQ ID NO: 1: a) codons 17, 30, 31, 44, 94, 134, 145, 165, 167, and 175, encoding for proline are each CCG; b) codons 16, 21, 25, 29, 35, 55, 57, 70, 115, 138, 161, 166, 185, 194, 216, 226, 255, 299, 302, and 310 encoding for threonine are each ACT; c) codons 19, 42, 76, 93, 118, 140, 147, 163, 197, 225, and 234 encoding for histidine are each CAC.

7. The polynucleotide of claim 1, wherein said nucleic acid sequence encoding for said protein comprises the sequence of SEQ ID NO: 3.42 4930-2481-6953.

18. The polynucleotide of any one of claims 1-6, wherein said nucleic acid sequence encoding said protein comprises a sequence at least 80% identical to any of SEQ ID NOs: 3-8.

9. The polynucleotide claim 8, wherein said nucleic acid sequence encoding said protein comprises a sequence at least 85% identical to any of SEQ ID NOs: 3-8.

10. The polynucleotide of claim 9, wherein said nucleic acid sequence encoding said protein comprises a sequence at least 90% identical to any of SEQ ID NOs: 3-8.

11. The polynucleotide of claim 10, wherein said nucleic acid sequence encoding said protein comprises a sequence at least 95% identical to any of SEQ ID NOs: 3-8.

12. The polynucleotide of claim 11, wherein said nucleic acid sequence encoding said protein comprises a sequence at least 97% identical to SEQ ID NO:

3.

13. A polynucleotide comprising a nucleic acid encoding for a protein comprising the amino acid sequence of SEQ ID NO: 9, wherein said polynucleotide comprises a nucleic acid sequence at least 90% identical to any of SEQ ID NOs: 3-8.

14. The polynucleotide of any one of claims of 1-13, wherein said nucleic acid sequence encodes a stop codon at the 3’ end of the SEQ ID NO: 9 encoding sequence.

15. The polynucleotide of any one of claims 1-14, wherein said polynucleotide is an expression cassette further comprises a promoter operatively linked to said nucleic acid sequence encoding said protein.

16. The polynucleotide of claim 15, further comprising a Shine-Dalgarno sequence operatively linked to said nucleic acid sequence encoding said protein.

17. The polynucleotide of claim 16, wherein said polynucleotide further comprises a polyadenylation sequence operatively linked to said nucleic acid sequence said encoding protein.

18. A plasmid comprising the polynucleotide of any one of claims 1-17, wherein said plasmid further comprises an origin of replication and a selectable marker.

19. A bacterial artificial chromosome or bacteriophage nucleic acid comprising the polynucleotide of any one of claims 1-17.

20. A recombinant prokaryotic cell comprising the polynucleotide of any one of claims 1-17, the plasmid of claim 18, or the bacterial artificial chromosome or the bacteriophage nucleic acid claim of 19.

21. A method of producing a protein comprising the sequence of SEQ ID NO: 9, wherein the method comprises culturing the recombinant prokaryotic cell of claim 20.

22. The method of claim 21, further comprising purifying said protein.

23. A method of preparing a pharmaceutical composition comprising a protein comprising the amino acid sequence of SEQ ID NO: 9, comprising the steps of: (a) producing said protein according to the method of claim 21;43 4930-2481-6953.1(b) purifying said protein to produce a purified protein; and (c) combining said purified protein with a pharmaceutically acceptable carrier.

24. A method of administering a viral vector to a subject comprising the steps of: (a) preparing a protein pharmaceutical composition comprising an amino acid sequence of SEQ ID NO: 9 according to the method of claim 23; (b) administering said protein pharmaceutical composition prepared in step (a) to the subject; and (c) administering the viral vector to the subject; wherein step (b) is performed prior to, after, or at the time as step (c).

25. The method of claim 24, wherein the viral vector is a recombinant adeno-associated viral vector comprising a nucleic acid encoding a therapeutic protein or a biologically active nucleic acid.

26. A method of treating an autoimmune disease in a subject comprising the steps of: (a) preparing a protein pharmaceutical composition comprising the amino acid sequence of SEQ ID NO: 9, according to the method of claim 23; and (b) administering the protein pharmaceutical composition prepared in step (a) to the subject.

27. A method of organ or tissue transplantation to a subject comprising the steps of: (a) preparing a protein pharmaceutical composition comprising the amino acid sequence of SEQ ID NO: 9, according to the method of claim 23; (b) administering the protein pharmaceutical composition prepared in step (a) to the subject; and (c) transferring the organ or tissue to the subject; wherein step (b) is performed prior to, after, or at the time as step (c).

28. The method of any one claims 24-27, wherein the subject is a human.44 4930-2481-6953.1

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