IDES fusion proteins

Abstract

The present invention features fusion proteins comprising an immunoglobulin G-degrading enzyme domain and an albumin binding domain. The albumin binding domain can bind to human serum albumin and can be useful, for example, for increasing the half-life of the immunoglobulin G-degrading enzyme domain in a subject. Uses of the fusion protein include treatment reducing a potential host immune response to an administered treatment such as those involving a viral vector, or involving a tissue or organ transplant; treating an adverse IgG immune response; and treating an autoimmune disease or disorder.

Description

Attorney Docket No. 065830.11549 / 20WO1IDES FUSION PROTEINSRELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No. 63 / 703,582, filed October 4, 2024, hereby incorporated by reference herein in its entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The contents of the electronic sequence listing (065830-20W01.xml; Size: 82,374 bytes; and Date of Creation: September 4, 2025) is herein incorporated 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 deliver, where IdeS is used to decrease IgG against the viral vector. (Bjbrck, J. Clin. Cell. Immunol. 2016, 7:2; U.S. Patent No. 10696959; Bou-Jaoudeh et al., Haematologica. 2023 Jan 19. doi: 10.3324 / haematol.2022.281895; Segelmark and Bjbrck, Front. Immunol. 2019, 10:2165, doi: 10.3389 / fimmu.2019.02165; International Patent Publication Nos.W02020 / 016318, WO2022 / 266044 and WO 2020 / 102740; Ros-Ganan et al., Clin. Transl. Immunology. 2022, Feb 24; 1 l(2):el375, and Leborgne et al., Nat. Med. 2020, 26(7): 1096- 1101.)

[0005] Another example for the use of IdeS is the production of F(ab’)2. (See, for example, 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 W02020 / 016318; and U.S. Patent Nos. 11,053,280, 10,696,959, and 7,666,582.4897-1312-5998.1BRIEF SUMMARY OF THE INVENTION

[0007] The present invention features fusion proteins comprising an immunoglobulin G- degrading enzyme domain and an albumin binding domain. The albumin binding domain can bind to human serum albumin and can be useful, for example, for increasing the half-life of the immunoglobulin G-degrading enzyme domain in a subject. Uses of the fusion protein include treatment reducing a potential host immune response to an administered treatment such as those involving a viral vector, or involving a tissue or organ transplant; treating an adverse IgG immune response; and treating an autoimmune disease or disorder.

[0008] Thus, a first aspect of the present invention is directed to a fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain. Unless otherwise indicated, the immunoglobulin G-degrading enzyme domain can be located in the fusion protein at the amino or carboxy side with respect to the human serum albumin binding domain. The two domains can be directly joined together or can be joined through a linker, such as an amino acid linker. Additional domains may be present at the amino and / or carboxy terminus of the fusion protein.

[0009] Another aspect of the present invention is directed to a polynucleotide comprising a sequence encoding a fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain.

[0010] Another aspect of the present invention is directed to an mRNA construct comprising a polynucleotide comprising a sequence encoding a fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain, wherein the polynucleotide is an RNA polynucleotide and the construct comprises: a 5’-cap, a 5’UTR, the RNA polynucleotide, a 3’UTR, and a poly(A) tail.

[0011] Another aspect of the present invention is directed to a recombinant cell comprising a polynucleotide encoding a fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain. The polynucleotide can be present, for example, in the recombinant cell chromosome or as an extrachromosomal element. In certain embodiments the recombinant cell is used to produce the fusion protein; and in a further embodiment, the fusion protein is purified from the recombinant cell.

[0012] Another aspect of the present invention is directed to a pharmaceutical composition comprising: (1) a fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain or (2) an mRNA construct encoding the fusion protein; and a pharmaceutically acceptable carrier. The mRNA construct can be provided, for example, within a non-viral gene delivery vehicle.

[0013] Another aspect of the present invention is directed to a method of decreasing IgG in a24897-1312-5998.1subject comprising administering to the subject a fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain or a mRNA construct encoding the fusion protein. In certain embodiments, the method is used to treat an autoimmune disease or immune-mediated condition in the subject; or used in conjugation with a tissue or organ transplant.

[0014] Another aspect of the present invention is directed to a method of administering a transgene to a subject comprising the steps of administering to the subject a viral vector comprising the transgene and either (1) a fusion protein comprising an immunoglobulin G- degrading enzyme domain and a human serum albumin binding domain or (2) an mRNA construct encoding the fusion protein. The mRNA construct encoding the fusion protein can be, delivered to a subject, for example, within a non-viral gene delivery vehicle.

[0015] Additional aspects of the present invention include a fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain and / or mRNA construct encoding for the fusion protein, for uses described herein; and in preparation of a medicament for medicine or for uses described herein.

[0016] 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

[0017] FIG. l is a bar diagram illustrating the ability of different IdeS variants to cleave IgG in pooled human plasma. Pooled human plasma was incubated with recombinant wild-type (WT) IdeS, recombinant IdeS variant, or vehicle alone (PBS). The molar quantity of enzyme was the same in all reactions. The relative quantity of enzyme (20 ng IdeS WT) added to plasma (18.5 pL) was low with the intent of partial IgG digest. Plasma samples were subsequently run on an automated Simple Western™ (Wes™) capillary Western blot system (biotechne / ProteinSimple) using a polyclonal anti-human IgG (heavy-chain + light-chain) detection antibody. The area of intact human IgG, single-cleaved IgG, and fully-cleaved F(ab’)2 histograms were quantified using Compass™ software (biotechne, ProteinSimple) and are represented as a percentage of total combined band area per capillary. Mean ± SD of N = 2 capillaries for wild type (WT) IdeS (SEQ ID NO: 2), IdeS-PK-1 (SEQ ID NO: 20), IdeS-PK-3 (SEQ ID NO: 21), IdeS-PK-4 (SEQ ID NO: 22), IdeS-PK-8 (SEQ ID NO: 23), IdeS-PK-9 (SEQ ID NO: 24).34897-1312-5998.1

[0018] FIGs. 2A-2F illustrate the ability of different albumin-binding IdeS variants to bind to native albumin from multiple species by using an ELISA. A 96-well plate was coated with purified native albumin from human (HSA), cynomolgus macaque (CSA), rabbit (RSA), or mouse (MSA). Serial dilutions of recombinant IdeS and IdeS variant proteins were added to each well, followed by detection with a goat anti-IdeS (anti-FabRICATOR®) primary antibody and a horseradish-peroxidase-conjugated secondary anti-goat-Fc antibody. Wells were developed with 3,3',5,5'-tetramethylbenzidine (TMB) substrate, stopped with 2 N sulfuric acid, and absorbance was measured at 450 nm wavelength. (Mean ± SD of duplicate ELISA wells.) FIG. 2A illustrates results with wild-type IdeS (SEQ ID NO: 2), FIG. 2B illustrates results with the construct IdeS-PK-1 (SEQ ID NO: 20), FIG. 2C illustrates results with the construct IdeS- PK-3 (SEQ ID NO: 21), FIG. 2D illustrates results the with construct IdeS-PK-4 (SEQ ID NO: 22), FIG. 2E illustrates results with the construct IdeS-PK-8 (SEQ ID NO: 23), and FIG. 2F illustrates results with the construct of IdeS-PK-9 (SEQ ID NO: 24).

[0019] FIGs. 3 A-3F illustrate one-phase decay of albumin-binding IdeS variants in sera of hFcRn+ / +, hAlb+ / + transgenic mice. Recombinant IdeS and IdeS variant (IdeS-PK-Seq) proteins were administered to dual-transgenic hFcRn+ / +, hAlb+ / + mice that express human neonatal Fc-receptor (FcRn) and human serum albumin under the control of their respective endogenous mouse promoters. Each animal (GN-XXX) received a single intravenous dose of IdeS or IdeS-variant protein. The relative decay and half-life of each IdeS variant in plasma was determined by serial blood collections and quantitative capillary electrophoretic Wes™ detection of IdeS in serum with an anti -IdeS antibody (anti-FabRICATOR®). The peak height (arbitrary units, AU) of each IdeS or IdeS-PK-Seq variant histogram was quantified using Compass™ software. Data points indicate IdeS or IdeS-PK-Seq variant Wes™ histogram peak heights as a function of time. A best-fit 1 -phase decay curve was fitted to data points for each individual animal. The dotted horizontal line in each panel represents the peak height mean + 3 standard deviations of 3 technical replicates of pooled sera from untreated animals (i.e. respective IdeS variant Wes™ assay “noise”). FIG. 3 A illustrates results with native IdeS (SEQ ID NO: 2), FIG. 3B illustrates results with the construct IdeS-PK-1 (SEQ ID NO: 20), FIG. 3C illustrates results with the construct IdeS-PK-3 (SEQ ID NO: 21), FIG. 3D illustrates results with the construct IdeS-PK-4 (SEQ ID NO: 22), FIG. 3E illustrates results with the construct IdeS-PK-8 (SEQ ID NO: 23), and FIG. 3F illustrates results with the construct IdeS-PK-9 (SEQ ID NO:24).

[0020] FIGs. 4A and 4B illustrate estimated half-life of albumin-binding IdeS variants and decline in hFcRn+ / +, hAlb+ / + transgenic mouse sera over time. Each animal received a single intravenous dose of IdeS or IdeS-variant protein. The relative decay and half-life of each IdeS44897-1312-5998.1variant in plasma was determined by serial blood collections and quantitative capillary electrophoretic Wes™ detection of IdeS in serum with an anti-IdeS antibody (anti- FabRICATOR®). FIG. 4A) A best-fit 1-phase decay curve of IdeS peak height versus elapsed time was used to determine the half-life of IdeS (WT) or albumin-binding IdeS variant (IdeS- PK). Data points represent the half-life determined for each animal, and bars indicate the mean ± SD half-life of n = 3 - 4 animals per group. A one-way ANOVA with Dunnett’s test for multiple comparisons was performed to determine statistical significance of mean half-life for each IdeS variant (IdeS-PK) relative to IdeS (WT) (*, p < 0.05, ****, p < 0.0001). FIG. 4B) For each animal, Wes™ peak height at each timepoint was normalized as a percentage of peak height at the first serum collection (approximately 30-minutes post-injection). Mean ± SD normalized peak height per group was plotted for each serum collection timepoint (horizontal line, 100%). Data for the following constructs are shown: native IdeS (SEQ ID NO: 2), IdeS-PK-1 (SEQ ID NO: 20), IdeS-PK-3 (SEQ ID NO: 21), IdeS-PK-4 (SEQ ID NO: 22), IdeS-PK-8 (SEQ ID NO: 23), and IdeS-PK-9 (SEQ ID NO: 24).DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention features fusion proteins comprising an immunoglobulin G- degrading enzyme domain and an albumin binding domain. The albumin binding domain can bind to human serum albumin and can be useful, for example, for increasing the half-life of the immunoglobulin G-degrading enzyme domain in a subject. Uses of the fusion protein include treatment reducing a potential host immune response to an administered treatment such as those involving a viral vector, or involving a tissue or organ transplant; treating an adverse IgG immune response; and treating an autoimmune disease or disorder.

[0022] 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 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 immunoglobulin G-degrading enzyme fusion protein described herein can be treated.

[0023] In certain embodiments, non-human subjects, cell cultures, eukaryotic cells or prokaryotic cells can be employed for producing an immunoglobulin G-degrading enzyme fusion protein.

[0024] Reference to an expression cassette indicates a sequence containing a transgene operably linked to one or more regulatory element providing for RNA expression from the transgene. The produced RNA can itself be functional or can encode for a protein. One type of regulatory54897-1312-5998.1element is a promoter, which binds RNA polymerase and the necessary transcription factors to initiate transcription. When encoding for 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 providing for expression. Additional regulatory elements include those impacting RNA expression, RNA stability, and protein production.

[0025] 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.

[0026] The term “operably linked” refers to the association of two or more nucleic acid segments on a single nucleic acid where the function of one is affected by the other.

[0027] Reference to “transgene” indicates a DNA region capable of being expressed to RNA, without regard to origin of the polynucleotide sequence. Transgenes encoding for protein can be operably linked to elements needed for protein expression, and is generally part of a recombinant nucleic acid, where the recombinant nucleic acid contains at least one region with which the transgene is not normally associated with in nature.

[0028] The singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

[0029] 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. 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 more than one of the options is also understood to fall within the meaning of the term “and / or.”

[0030] 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 member or examples can be provided or used.

[0031] 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.

[0032] 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 additional64897-1312-5998.1unrecited elements or method steps. Terms such as “including”, “containing” and “characterized by” 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’.

[0033] 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.

[0034] 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.

[0035] 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%.

[0036] 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.

[0037] 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, 15 or more; and administration “two or more” times includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more times.

[0038] 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. In determining percent identity for a polynucleotide, RNA and the corresponding DNA are considered the same unless provided otherwise by the employed context, for example, providing reference to the polynucleotide being74897-1312-5998.1RNA or DNA. Corresponding RNA and DNA include uracil for thymine and replacement of the ribose backbone for the deoxyribose backbone.

[0039] 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 a polypeptide of SEQ ID NO: 1 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: 1 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.

[0040] Reference to differing with respect to a reference sequence (e.g., an amino acid or nucleic acid sequence), provides for the indicated number of differences, where each difference is independently a substitution, deletion, or addition. In certain embodiments for an amino acid sequence, each difference is a substitution; and a further embodiment each substitution is a conservative substitution.

[0041] Various references including articles and patent publications are cited or described in the background and throughout the specification. Each of these references is 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.

[0042] The definitions provided herein, including those in the present section and other sections of the application apply throughout the present application.

[0043] 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.

[0044] 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 embodiments from the substance of another paragraph or section or embodiment. The provided descriptions have broad application and encompasses 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 clams), is limited to these examples.84897-1312-5998.1I. Fusion Protein

[0045] Featured fusion protein described herein comprise an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain. Within the fusion protein the immunoglobulin G-degrading enzyme domain and the human serum albumin binding domain can have different locations (e.g., either domain can be amino or carboxy to the other domain) and, optionally, additional amino acid regions may be present between the immunoglobulin G- degrading enzyme domain and human serum albumin binding domain, and / or at the amino or carboxy end of the immunoglobulin G-degrading enzyme domain and / or human serum albumin binding domain.

[0046] In certain embodiments, the fusion protein comprises the configuration, amino to carboxy: ABS-L-IDE, where ABS is the domain that binds human serum albumin, L is an optional first amino acid linker, and IDE is the immunoglobulin G-degrading enzyme domain, where the amino acid linker comprises at least 1 amino acid.

[0047] In certain embodiments, no linker is present and the fusion protein comprises the configuration, amino to carboxy: ABS-IDE, where ABS is the domain that binds human serum albumin and IDE is the immunoglobulin G-degrading enzyme domain.

[0048] In certain embodiments, the fusion protein comprises the configuration, amino to carboxy IDE-L-ABS, where ABS is the domain that binds human serum albumin, L is an optional first amino acid linker, and IDE is the immunoglobulin G-degrading enzyme domain, where the first amino acid linker comprises at least 1 amino acid.

[0049] In further embodiments, the first linker comprises 2, 3, 4, 5, 10, 20, 30, 40, 50, 60 or 70 amino acids

[0050] In certain embodiments the first amino linker comprises up to 10, 20, 30, 40, 50, 60 or 70 amino acids and comprises an amino acid sequence of any of SEQ ID NOs: 8-12; a sequence at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the sequence of any of SEQ ID NOs: 8-12; or a sequence differing from any of SEQ ID NOs: 8-12 by 1, 2, 3, 4, or 5 amino acids.

[0051] In certain embodiments, the fusion protein comprises one or more additional amino acid regions. In certain embodiments, the additional amino acid region is a linker. In further embodiments, a linker comprises a purification tag or a cleavage site. Purification tags, such as a His-tag can be used to facilitate purification. A protease cleavage tag can be used to facilitate separation of domains or regions, such as removing a purification 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. In certain embodiments the cleavage cite is provided by ENLYFQS (SEQ ID NO: 71).94897-1312-5998.1

[0052] In different embodiments, linkers are selected to provide for distance or flexibility between two regions or domains and / or provide a protease cleavage site between two regions.

[0053] In certain embodiments, nucleic acid encoding fusion protein comprises codons providing for a translational ramp to enhance translation initiation and / or encodes N-terminal amino acids comprising a translation initiation methionine.

[0054] In certain embodiments, the fusion protein (1) comprises an amino acid sequence of any of SEQ ID NOs: 15-24; (2) comprises an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the sequence of any of SEQ ID NOs: 15-24;(3) comprises an amino acid sequence differing from any of SEQ ID NOs: 15-24 by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; or (4) is a variant of a sequence provided in (1), (2) or (3), wherein an N-terminal methionine provided for in SEQ ID NOs: 15-24 is removed, for example, a variant of any of SEQ ID NOs: 15-24 lacking the N-terminal methionine.

[0055] Examples of different regions / domains present in SEQ ID NOs: 15-19 are characterized in Table 1, where MA refers to encoded N-terminal methionine-alanine.

[0056] Table 1

[0057] Fusion proteins of SEQ ID NOs: 20-24 were used to evaluate different human serum albumin binding domains, different linkers, and different orientations. SEQ ID NO: 20 comprises the same human serum albumin binding domain and immunoglobulin G-degrading enzyme domain sequences as SEQ ID NO: 15. SEQ ID NO: 21 comprise the same human serum albumin binding domain and immunoglobulin G-degrading enzyme domain sequence as SEQ ID NO: 16. SEQ ID NO: 22 comprises the same human serum albumin binding domain and immunoglobulin G-degrading enzyme domain as SEQ ID NO: 17. SEQ ID NO: 23 comprises the same human serum albumin binding domain and immunoglobulin G-degrading enzyme104897-1312-5998.1domain as SEQ ID NO: 18. SEQ ID NO: 24 comprises the same human serum albumin binding domain and immunoglobulin G-degrading enzyme domain as SEQ ID NO: 19.

[0058] In certain embodiments, the fusion protein comprises an amino acid sequence of SEQ ID NO: 18; or a sequence at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the sequence SEQ ID NO: 18; or differing from SEQ ID NO: 18 by 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acids.

[0059] In certain embodiments, the fusion protein comprises an amino acid sequence of SEQ ID NO: 19 or an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 19; or differing from SEQ ID NO: 19 by 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 amino acids.I, A, Immunoglobulin G-degrading Enzyme Domain

[0060] An immunoglobulin G-degrading enzyme domain comprises a sequence able to cleave IgG. Immunoglobulin G-degrading enzyme domains can be based, for example, on immunoglobulin G-degrading enzyme produced in a Streptococcus. The immunoglobulin G- degrading enzyme can be based on IdeS or other immunoglobulin G-degrading enzyme produced in a Streptococcus such as EndoS or IdeSORK (see, for example, International Patent Publication Nos. WO2022 / 266044, W02020 / 016318, and WO2022 / 223818 each of which are incorporated by reference herein in their entirety); and U.S. Patent Nos. 11,053,280, 10,696,959, and 7,666,582; each of which are hereby incorporated by reference herein in their entirety).

[0061] U.S. Patent No. 10,696,959 (incorporated by reference herein in its entirety) describes different amino acids important for IdeS activity and provides examples of different alterations (additions, substitutions, and / or deletions) that can be made and still retain activity. An important residue for IgG cysteine protease activity is indicated to be cysteine (C) at a position corresponding to amino acid 65 of SEQ ID NO: 2 (mature IdeS). Other residues indicated likely to be important for IgG cysteine protease activity are the lysine (K) at position 55, the histidine (H) at position 233, and the aspartic acid (D) at each of positions 255 and 257, each with respect to SEQ ID NO: 2.

[0062] U.S. Patent No. 10,696,959 (incorporated by reference herein in its entirety) at Table C provides examples of different types of IdeS modifications. In certain embodiments, the immunoglobulin G-degrading enzyme domain comprise one or more modification provided in U.S. Patent No. 10,696,959 Table C. In further embodiments the immunoglobulin G-degrading enzyme domain has one or more modification corresponding to (with respect to SEQ ID NO: 1): K115E; E119K; N130K or N130R; E139K; D142R; E198R or E198K; D216N; K241S or K241E; T244D or T244E; E245N or E245K; S302K; D316K; and / or D333K.114897-1312-5998.1

[0063] In certain embodiments, the immunoglobulin G-degrading enzyme domain comprises a cysteine (C) at a position corresponding to amino acid 94 of SEQ ID NO: 2. In further embodiments, the immunoglobulin G-degrading enzyme domain with respect to SEQ ID NO: 2, has a lysine (K) at a position corresponding to amino acid 84 of SEQ ID NO: 2; a histidine (H) at a position corresponding to amino acid 262 of SEQ ID NO: 2; an aspartic acid (D) at a position corresponding to amino acid 284 of SEQ ID NO: 2; and an aspartic acid (D) at a position corresponding to amino acid 286.

[0064] U.S. Patent No. 10,696,959 (incorporated by reference herein in its entirety) describes particular IdeS variants indicated to provide for immunoglobulin G-degrading activity. In certain embodiments, the immunoglobulin G-degrading domain comprises an IdeS variant described in U.S. Patent No. 10,696,959, or a sequence having a sequence identity of at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or differs from a described variant in U.S. Patent No. 10,696,959 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids to such a domain.

[0065] SEQ ID NOs: 25-38 of the present application correspond to different sequence provided in U.S. Patent No. 10,696,959. In certain embodiments, the immunoglobulin G-degrading domain comprises a sequence of any of SEQ ID NOs: 25-38, or a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, to any of SEQ ID NOs: 25-38, or a sequence differing from any of SEQ ID NOs: 25-38 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.

[0066] International Patent Publication No. WO2022 / 266044 (incorporated by reference herein in its entirety) describes different IdeS variant sequences having functional activity. Examples of IdeS variants described in WO2022 / 266044 include those designated mt5, mt6, mt8, mt9 and mtlO corresponding to SEQ ID NOs: 39-43 of the present application. WO2022 / 266044 mt5, mt6, mt8, mt9 and mtlO sequences include the signal sequence, without the N-terminal methionine. In certain embodiments, the immunoglobulin degrading domain comprises the mature sequence provided by any of SEQ ID NOs: 39-43, or a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, to any of SEQ ID NOs: 39-43, or a sequence differing from any of SEQ ID NOs: 39-43 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.

[0067] International Patent Publication No. WO2022 / 223818 (incorporated by reference herein in its entirety), describes a polypeptide having immunoglobulin protease activity, referenced in the patent publication as IdeSORK, along with a shorter fragment providing for immunoglobulin protease activity. In certain embodiments, the immunoglobulin G-degrading enzyme domain comprises a sequence provided by SEQ ID NOs: 44 or 45, or a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at124897-1312-5998.1least 98%, at least 99%, to SEQ ID NOs: 44 or 45, or a sequence differing from SEQ ID NOs: 44 or 45 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.

[0068] US Patent No. 11,053,280 (incorporated by reference herein in its entirety) describes different IdeS variant sequences indicated to provide immunoglobulin degrading activity. The sequence designated FabRICATOR is indicated to include asparagine residues at position 87, 130, 182 and / or 274, that can be mutated to a different amino acid. SEQ ID NOs: 46-61 described herein, correspond to different sequences provided in US Patent No. 11,053,280. In certain embodiments, the immunoglobulin degrading domain comprises the sequence provided by any of SEQ ID NOs: 46-61, or a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, to any of SEQ ID NOs: 46-61, or a sequence differing from any of SEQ ID NOs: 46-61 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.LB. Human Serum Albumin Binding Domain

[0069] A variety of different types of polypeptide domains can bind to human serum albumin including, for example, domains generated from albumin-binding prokaryotic proteins, domains identified from synthetic polypeptide screening, and domains derived from engineered antibody binding regions. In certain embodiments the human serum albumin binding domain can bind to human serum albumin of SEQ ID NO: 13 in vitro with nanomolar or better affinity, using for example, assays provided for in the Examples below.

[0070] In certain embodiments the human serum albumin binding domain comprises an antibody variable region. Examples of antibody variable regions include those based on vertebrate heavy chain antibody variable regions, such as those found in multi-chain antibodies and those found in single-chain antibodies. Vertebrates antibodies include those produced by mammals and non-mammals. Examples of naturally occurring single-chain antibodies include immunoglobulin new antigen receptor (IgNAR), present in, for example, cartilaginous fish; and nanobodies (heavy chain variable regions) present in, for example, camelidae (see, for example, Fernandez-Quintero et al., Front Mol Biosci. 2021 Apr 20;8 and Muller et al., MAbs. 2012 Nov- Dec;4(6):673-685).

[0071] IgNAR and mammalian antibody variable regions differ in the number of complementary determining regions binding to an epitope. Mammalian antibody variable regions comprise three complementary determining regions (CDR1, CDR2 and CDR3) able to bind an epitope, interspaced within a framework. IgNAR comprise two complementary determining regions binding to an epitope (see, for example, Fernandez-Quintero et al., Front Mol Biosci. 2021 Apr 20;8 and Muller et al., MAbs. 2012 Nov-Dec;4(6):673-685).134897-1312-5998.1

[0072] Human domain antibody variable regions targeting human serum albumin are illustrated, for example, in Holt et al., Protein Eng Des Sei. 2008 May;21(5):283-288 (Holt et al.) Walker et al., Protein Engineering, Design and Selection, Volume 23, Issue 4, April 2010, pages 271-278 (Walker et al.,), and Bao et al., Cardiovasc Diabetol 12, 148 (2013) each of which are hereby incorporated by reference herein in their entirety. Holt et al. and Walker et al., include descriptions of different CDRs combinations (CDR 1, 2, and 3) present in domain antibodies targeting human serum albumin. In certain embodiments, the domain antibody targeting human serum albumin comprise CDR combinations identified in Holt et al. or Walker et al. In a further embodiment, the CDR combination is that present in Dom7h-14 (see Walker et al.) wherein CDR1 is provided by RASQWIGSQLS (SEQ ID NO: 62), CDR2 is provided by WRSSLQS (SEQ ID NO: 63), and CDR3 is provided by AQGAALPRT (SEQ ID NO: 64).

[0073] Shark domain antibodies targeting human serum albumin are illustrated, for example, in Muller et al., MAbs. 2012 Nov-Dec;4(6):673-685 (Muller), hereby incorporated by reference herein in its entirety. Muller includes descriptions of different CDRs present in domain antibodies targeting human serum albumin. In certain embodiments, the domain antibody targeting human serum albumin comprise CDR combinations identified in Muller. In a further embodiment, the CDR combination is that present in E06, wherein CDR1 is provided by SYPLYS (SEQ ID NO: 65) and CDR3 is provided by MGTNIWTGDGA (SEQ ID NO: 66).

[0074] In certain embodiments, the human serum albumin binding domain comprises a polypeptide that does not contain a CDR. Techniques for producing such polypeptides, are illustrated, for example, in Dennis et al., J. Biol. Chem. 2002 Sep 20;277(38):35035-43 hereby incorporated by reference herein in its entirety. In certain embodiments, the polypeptide binding to human serum albumin comprises a core sequence of DICLPRWGCLW (SEQ ID NO: 14) or the sequence of SEQ ID NO: 5.

[0075] Additional examples of albumin binding polypeptides are described in Kraulis et al., FEBS Letters, Volume 378, Issue 2, 1996, pages 190-194 and Jonsson et al., Protein Eng Des Sei. 2008 Aug;21(8):515-27 (both of which are hereby incorporated by reference herein in their entirety). In certain embodiments the albumin binding domain comprises the amino acid sequence of SEQ ID NOs: 3 or 4.II. Fusion Protein Production and Purification

[0076] A fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding can be recombinantly expressed and purified. Recombinant expression can be achieved using recombinant nucleic acid encoding for the enzyme. The recombinant nucleic acid can be part of the host chromosome or exist as an extracellular144897-1312-5998.1element. Reference to “recombinant” refers to one or more components not occurring in nature and / or a combination of components not occurring in nature.II. A, Prokaryotic Expression Cassettes

[0077] In certain embodiments expression is carried out in a prokaryotic host from an expression cassette suitable for prokaryotic expression. The expression cassette comprises a transgene encoding a fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding, operably linked to one or more regulatory and expression control elements needed for RNA production, processing and translation. Examples of prokaryotic regulatory and expression control elements include a promoter, an enhancer, and a Shine- Dalgarno sequence.

[0078] In certain embodiments the expression cassette is present on an expression vector, such as a plasmid that replicates independently of the host chromosome.

[0079] In certain embodiments, the expression cassette is present in the prokaryotic host genome.

[0080] A promoter contains a DNA region where transcription is initiated. 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.

[0081] 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, T71ac, 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 Rincon, 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 nucleic acid encoding for the fusion construct comprises a Lac promoter and Lac operon. In certain embodiments, the promoter is a lac promoter, and isopropyl-P-D-thiogalactoside (IPTG) is used for induction.

[0082] In certain embodiments, the polynucleotide comprises a Shine-Dalgamo sequence providing a ribosomal binding site and facilitating RNA translation. (See, for example, Wen et al., RNA Biol. 2021 Nov;18(l l): 1489-1500, hereby incorporated by reference herein in its entirety.)154897-1312-5998.1

[0083] 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, such as mRNA turnover. (Hajnsdorf and Kaberdin, 2018 Phil. Trans. R. Soc. B 373: 20180166, hereby incorporated by reference herein in its entirety.)

[0084] Plasmids can be used as extrachromosomal elements and / or for insertion of DNA into a host chromosomal. In addition to an expression cassette comprising a sequence encoding a transgene along with regulatory and expression control elements, a plasmid typically comprises additional elements such as those providing for plasmid replication and a selectable marker.

[0085] 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 genes providing an enhanced ability to utilize certain nutrients such as particular amino acids.

[0086] In certain embodiments the transgene encoding an immunoglobulin G-degrading enzyme domain and a human serum albumin binding is present in a bacteriophage nucleic acid or a bacterial artificial chromosomal. The bacterial artificial chromosomal comprises elements needed for replication and cell maintenance. The bacteriophage nucleic acid comprises elements for packaging into a bacteriophage.II.B, Production and Purification

[0087] Recombinant expression can be carried out in a prokaryotic host. 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.

[0088] Prokaryotic cells 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.

[0089] 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; and in International Patent Publication Nos. WO2022 / 266044 and W02020 / 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,164897-1312-5998.1

[0090] 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 cell host protein. In further embodiments, purification comprises the use of reverse phase liquid chromatography, size exclusion chromatography, and / or CE-SEC reduced chromatography.III, mRNA

[0091] RNA versions of nucleic acid encoding for a fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain, or other type of protein described herein, can be directly administered as an mRNA construct to a subject. The mRNA construct is able to express the encoded polypeptide inside a cell. The mRNA construct comprises a 5’-cap, a 5’UTR, the encoding RNA, 3’UTR, and a poly(A) tail. The UTRs and poly(a) tail can provide for different functions such as participating in mRNA subcellular localization, regulating translation efficiency and mRNA stability. The design and production of mRNA constructs, including different modifications, are illustrated in different publications such as Ouranidis et al., (2022) Biomedicines, 10, 50; Qin et al., Signal Transduct Target Ther. (2022) 21;7(I): 166, and U.S. Patent Publication No. U.S. 2013 / 0259924, each of which are hereby incorporated by reference herein in their entirety.

[0092] In certain embodiments, the mRNA construct is delivered to a cell or subject using nanoparticles. Examples of nanoparticles include those provided in Section VI infra., Ouranidis et al., (2022) Biomedicines, 10, 50, and U.S. Patent Publication No. U.S. 2013 / 0259924.IV. Gene Therapy

[0093] In certain embodiments, the fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain, or encoding mRNA construct is used in conjunction with gene therapy, for example, to decrease an immune response to a vector comprising a transgene. Gene therapy includes both loss-of-function and gain-of-function genetic defects. 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 normally associated with the wild-type protein.

[0094] 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. A gain-of-function mutation can be a deletion, addition, or substitution of a nucleotide or nucleotides in the gene, giving rise to a 174897-1312-5998.1change in the encoded protein function. In certain embodiments, the gain-of-function mutation changes the function of the mutant protein, causes interaction with other protein, or alters 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.

[0095] A transgene be used to produce a protein having a desired activity and / or a 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), or a transcription activator-like effector nuclease (TALEN).IV. A, Expression Cassettes for Gene Therapy

[0096] Polynucleotide expression cassettes contain a nucleic acid encoding for a polypeptide or a biologically active nucleic acid operably linked to one or more expression control elements. Expression control can be affected, for example, at the level of transcription, translation, splicing, and message stability. Expression control elements are typically located 5’ (“upstream”) or 3’ (“downstream”) of a transcribed nucleic acid. Expression control elements can also be located within the transcript (e.g., in an intron), adjacent to or at a distance away from the transcribed sequence. One or more expression control elements of the same or different type may be present. Examples of expression control elements for expression in a subject include a promoter, enhancer, an intron, polyadenylation signal, a Kozak sequence, post- transcriptional regulator elements and a termination sequence.

[0097] Expression control elements such a promoter and an enhancer can be chosen to preferentially drive expression in a particular cell or tissue type. Expression control elements are typically active in particular cells, tissues or organs because they are recognized by transcriptional activator proteins, or other regulators of transcription, that are unique to a specific cell, tissue or organ type. (See, e.g., Green, M. and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual. 4th Edition, Vol. II, Cold Spring Harbor Laboratory' Press, New York; and Ausubel et al., (2010) Current protocols in molecular biology, John Wiley & Sons, New York.)

[0098] The incorporation of tissue specific regulatory elements provides for at least partial tissue tropism for a desired protein being produced. Reference to a promoter or enhancer specific for a particular cell type or tissue, indicates the promoter or enhancer provides higher levels of expression and / or secretion in the indicated cell or tissue type. Examples of promoters specific for liver are the transthyretin (TTR) gene promoter; human alpha 1 -antitrypsin (hAAT) promoter; the apolipoprotein A-I promoter; albumin promoter, Miyatake et al., J. Virol.,184897-1312-5998.171 :5124-32 (1997); hepatitis B virus core promoter, Sandig et al., Gene Ther. 3: 1002-9 (1996); alpha-fetoprotein (AFP), Arbuthnot, et al., Hum. Gene. Ther., 7:1503-14 (1996); human Factor IX promoter; thyroxin binding globulin (TBG) promoter; TTR minimal enhancer / promoter; alpha-antitrypsin promoter; LSP (845 nt); and LSP1 promoter. An example of an enhancer active in liver is apolipoprotein E (apoE) HCR-1 and HCR-2 (Allan et al., J. Biol. Chem., 272:29113-19 (1997)).

[0099] Expression control elements also include ubiquitous or promiscuous promotors and promoters / enhancers capable of driving polynucleotide expression in many different cell types. Such elements include the EFl -alpha promoter, the cytomegalovirus (CMV) immediate early promoter / enhancer sequences, the Rous sarcoma virus (RSV) promoter / enhancer sequences, phosphoglycerate kinase (PKG) promoter, CAG (composite of the CMV enhancer, the chicken beta actin promoter (CBA) and the rabbit beta globin intron) (see, e.g., Boshart et al., (1985) Cell, 41 :521-530), the SV40 promoter, the dihydrofolate reductase promoter, and the cytoplasmic b-actin promoter.

[0100] Examples of CNS specific promoters include: neuron specific promoters such as the NSE (neuronal specific enolase), synapsin or NeuN, platelet-derived growth factor (PDGF), platelet- derived growth factor B-chain (PDGF-P), methyl-CpG binding protein 2 (MeCP2), Ca2 / calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), neurofilament light (NFL) or heavy (NFH), P-globin minigene nP2, preproenkephalin (PPE), enkephalin (Enk), and excitatory amino acid transporter 2 (EAAT2) promoters; astrocyte specific promoters such as the glial fibrillar acidic protein (GFAP) and EAAT2 promoters; oligodendrocyte specific promoters such as the myelin basic protein (MBP) / myelin-associated glycoprotein and oligodendrocyte transcription factor 2 promoter; neurons / hypothalamus specific promoters such as the proopiomelanocortin (POMC) promoter; and neuron / spinal cord specific promoter such as superoxide dismutase 1 (SOD1). (See, e.g., U.S. Patent Publication No. 2021 / 214749 and Adeno- Associated Virus Vectors (2019), Ed. Castle., 1stEdition, Springer New York, N.Y.; both of which are hereby incorporated by reference herein in their entirety.)

[0101] Additional promoters include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), herpes simplex virus (HSV) promoter, SFFV promoter, rat insulin promoter, TBG promoter, the desmin promoter and similar muscle-specific promoters, synthetic promoters, hybrid promoters, and promoters with multi-tissue specificity.

[0102] Expression control elements also can impact expression in a manner that is regulatable by a signal or stimuli increasing or decreasing expression. A regulatable element increasing194897-1312-5998.1expression of transcribed nucleic acid in response to a signal or stimuli is also referred to as an “inducible element” (i.e., is induced by a signal). Typically, the amount of increase or decrease conferred by such elements is proportional to the amount of signal or stimuli present. Particular examples include zinc-inducible sheep metallothionine (MT) promoter; the steroid hormone- inducible mouse mammary tumor virus (MMTV) promoter; the tetracycline-repressible system (Gossen, et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)); the tetracycline-inducible system (Gossen et al., Science 268: 1766-1769 (1995); see also Harvey et al., Curr. Opin. Chem. Biol. 2:512-518 (1998)); the RU486-inducible system (Wang et al., Nat. Biotech. 15:239-243 (1997) and Wang et al., Gene Ther. 4:432-441 (1997); and the rapamycin-inducible system (Magari et al., J. Clin. Invest. 100:2865-2872 (1997); and Rivera et al., Nat. Medicine. 2:1028- 1032 (1996)). Other examples of regulatable control elements include those regulated by a specific physiological state such as temperature, acute phase, or development.

[0103] In certain embodiments the expression cassette further comprises one or more introns independent of a polypeptide encoding nucleotide sequence. A variety of different introns can be used to enhance gene expression. Examples of introns that may be used include the rabbit P- globin intron with splice donor / splice acceptor, SV40 intron with splice donor / splice acceptor, human P-globin introns, intron 2 of the human hemoglobin beta gene, hFIX inti (intron 1 of the human coagulation factor IX gene), CBA-rHHB (synthetic intron derived from the fusion of the intron 1 of the chicken beta actin gene and intron 2 of the rabbit hemoglobin beta), CBA (intron 1 of the chicken beta actin gene), hGH (intron 1 of the human growth hormone gene), hFIX synthetic (synthetic intron derived from different portions of the human coagulation factor IX gene and present in the pLIVE vector, Minis Bio, Madison, WI); human hemoglobin subunit beta (HBB2) synthetic intron, and optimized HBB2; and chimeric introns such as introns made up of the 5 '-splice donor of the first human P-globin intron and the branch and 3 '-acceptor site from the intron that is between the leader and the body of the immunoglobulin gene heavy chain variable region. (Buck et al., Int. J. Mol. Sci. (2020), 21, 4197; Ronzitti et al., Mol. Ther.Methods Clin Dev. (2016) Jul 20;3 : 16049; and the HBB-IGG intron provided by the pCMVNT™ vector.)

[0104] In certain embodiments the expression cassette comprises a post-transcriptional regulatory element. Post-translational regulatory elements such as Woodchuck post- transcriptional regulatory element (WPRE) and Hepatitis B regulatory element can increase gene expression. (Buck et al., Int. J. Mol. Sci. (2020), 21, 4197.)

[0105] Polyadenylation signal sequences provide for the formation of a polyA tail, which facilitates nuclear export, translation and / or mRNA stability, and may also be involved in transcription termination. Examples of polyadenylation signal sequences include SV40 late204897-1312-5998.1polyadenylation signal, bovine growth hormone polyA (bGHpA) signal sequence, synthetic poly A, mouse P-globin pA, rabbit P-globin pA, and H4-based pA. (Buck et al., Int. J. Mol. Sci. (2020), 21, 4197.)

[0106] In certain embodiments, the expression cassette comprises a Kozak consensus sequence or a variation thereof. Kozak consensus sequences play a role in translation initiation. The Kozak consensus sequence and variations are provided in, for example, McClements et al., (20 1) Molecular vision, 27, 233-242, hereby incorporated by reference herein,

[0107] In certain embodiments the expression cassette further comprises a miRNA target sequence, which in further embodiments is incorporated into the 3’ UTR of the expression cassette. A miRNA target sequence is recognized by miRNA present in particular cells or tissues leading to degradation of mRNA transcripts. Based on the presence of certain miRNA in particular cells, incorporating a miRNA target sequence(s) can be used to reduce expression in certain cells or tissue types. Multiple tandem repeats of miRNA target sequences can be used to increase degradation. (Geisle et al ., (2016) World Journal of Experimental Medicine 6(2): 37- 54.)

[0108] In certain embodiments, nucleic acid encoding for a polypeptide comprises a secretory signal sequence to facilitate extracellular secretion of the polypeptide. The term “secretory signal sequence” refers to amino acid sequences functioning to enhance secretion of an operably linked polypeptide from the cell as compared to the level of secretion seen with the polypeptide lacking the secretory signal sequence. It is not necessary that essentially all or even most of the polypeptide is secreted, as long as the secretion level is enhanced as compared with the native polypeptide. Generally, secretory signal sequences are cleaved within the endoplasmic reticulum and may be cleaved prior to secretion. It is not necessary that the secretory signal sequence is cleaved as long as secretion of the polypeptide from the cell is enhanced and the polypeptide is functional.

[0109] The secretory signal sequence can be derived in whole or in part from the secretory signal of a secreted polypeptide (e.g., from the precursor) and / or can be in whole or in part synthetic. The length of the secretory signal sequence is not critical, and can be for example, from about 10-15 to 50-60 amino acids in length. Known secretory signals from secreted polypeptides can be altered or modified (e.g., by substitution, deletion, truncation, or insertion of amino acids) as long as the resulting secretory signal sequence functions to enhance secretion of an operably linked polypeptide. The secretory signal sequences can comprise, consist essentially of, or consist of a naturally occurring secretory signal sequence or a modification thereof. Examples of synthetic or artificial secretory signal peptides are provided in Barash et al.,Biochem. Biophys. Res. Comm. 294, 835 (2002).214897-1312-5998.1

[0110] In certain embodiments, the expression cassette nucleotide sequence contains any of 0-5, 0-10, 0-15, 0-50, or 0-100 CpGs; 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 CpGs; 0%, about 0.5%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% CpGs; and / or up to about 0.5%, up to about 1.0%, up to about 2.0%, up to about 3.0%, up to about 4.0%, up to about 5.0%, up to about 6%, up to about 7%, up to about 8%, up to about 9%, up to about 10%, up to about 11%, up to about 12%, up to about 13%, up to about 14%, or up to about 15% CpGs.IV.B, Therapeutic Proteins[OHl] In certain embodiments the transgene construct encodes for a protein having a desired activity. Examples of transgenes include those providing a healthy copy of a gene in a subject where the gene is defective, a modified gene that can help treat a disease or disorder, or a new gene encoding for protein providing a beneficial effect.

[0112] In different embodiments, a 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 (Cl protease inhibitor or Cl esterase inhibitor) for treatment of hereditary angioedema (HAE), also known as Cl inhibitor deficiency type I and type II); or glucose-6- phosphatase for treatment of glycogen storage disease type I (GSDI).

[0113] 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), angiopoietins, 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 a (TGFa), platelet-derived growth factor (PDGF), insulin growth factors I or II (IGF-I or IGF-II), TGFP, activins, 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), ephrins, noggin, sonic hedgehog or tyrosine hydroxylase.224897-1312-5998.1

[0114] 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 a or P, interferons a, P, or y, stem cell factor, flk-2 / flt3 ligand, IgG, IgM, IgA, IgD or IgE, chimeric immunoglobulins, an antibody, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, class I or class II MHC molecules. Antibodies and immunoglobulins can, for example, be provided targeting cancer cells or other disease or disorder causing cells.

[0115] 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 Vila, or protein C) a gain of function blood coagulation factor, erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, P-globin, a-globin, spectrin, a-antitrypsin, adenosine deaminase (ADA), a metal transporter (ATP7A or ATP7), sulfamidase, an enzyme involved in lysosomal storage disease (ARSA), hypoxanthine guanine phosphoribosyl transferase, P-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 a and P, a cytokine, a-interferon, P-interferon, interferon-y, interleukin-2, interleukin-4, interleukin 12, granulocyte-macrophage colony stimulating factor, lymphotoxin, a suicide gene product, herpes simplex virus thymidine kinase, cytosine 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), adenomatous polyposis coli (APC)), a peptide with immunomodulatory properties, a tolerogenic or immunogenic peptide or protein Tregitope or hCDRl, 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-acetyl glucosamine- 1 -phosphate transferase, cathepsin A,234897-1312-5998.1GM2-AP, NPC1, VPC2, a sphingolipid activator protein, or one or more donor sequences used as repair templates for genome editing.

[0116] In different embodiments, the transgene construct encodes erythropoietin (EPO) for treatment of anemia; interferon-alpha, interferon-beta, and 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; alphal -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; apolipoproteins 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; argininosuccinate synthetase; 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.

[0117] In a further embodiment 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, wet macular degeneration, Leber hereditary optic neuropathy, and Stargardt disease.IV.C. Biologically Active Nucleic Acid

[0118] Biologically activity nucleic acid can alter the activity of other nucleic acid. Nucleic acid244897-1312-5998.1encoding the biologically active nucleic acid can be delivered to a subject using, for example, a viral vector. In certain embodiments, a fusion protein comprising an immunoglobulin G- degrading enzyme domain and a human serum albumin binding domain is used in conjunction with a viral vector comprising nucleic acid encoding a biologically active nucleic acid.

[0119] In certain embodiments, the biologically activity nucleic acid is an inhibitory nucleic acid effecting transcription or translation. Examples of inhibitory nucleic include a short hairpin RNA (shRNA), a small interfering RNA (siRNA), a microRNA (miRNA), a ribozyme, and an antisense RNA.

[0120] In certain embodiments, the biologically active nucleic acid is a small activating RNA (saRNA), which can increase transcription. (See, for example, Tan et al., Gene Transcription. Molecules. 2021 Oct 28;26(21):6530, hereby incorporated by reference herein in its entirety.)

[0121] 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 selected from the group consisting of 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 (CACNA1 A), 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, 12 17), FMRI (fragile X mental retardation 1) in fragile X syndrome, FMRI (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) and proprotein convertase subtilisin / kexin type 9 (PCSK9), 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), in metastatic melanoma; LMP7, also known as proteasome subunit beta-type 8 (PSMB 8), in metastatic melanoma; MECL1 also known as proteasome subunit beta-type 10 (PSMB 10), in metastatic melanoma; vascular endothelial growth factor (VEGF) in solid tumors; kinesin spindle protein254897-1312-5998.1in 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, in glaucoma; RTP801 / Reddl 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 (TORI A) 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.D. Gene Editing

[0122] In certain embodiments, a fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain, is used in conjunction with gene editing nucleic acid or protein. Examples of gene editing nucleic acid and protein include ZFN, TALEN, and CRISPR-Cas9. In different embodiments the gene editing nucleic acid or protein edits a subject’s DNA to provide a therapeutic protein as provided in Section IV.B. supra., or disrupt a gene as provided in Section IV.C. supra.V. Viral Vectors

[0123] In certain embodiments, the fusion protein comprising an immunoglobulin G-degrading enzyme domain is used in conjugation with a viral vector in a subject. A viral vector comprises recombinant viral nucleic acid encapsidated in a protein capsid. The viral vector can deliver the viral vector 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 transgene delivery include adenovirus vectors, rAAV, retrovirus vectors and herpes simplex vectors.

[0124] Recombinant viral vector nucleic acid contains 5’ and / or 3’ viral elements providing for viral packaging and may provide for additional activities such as self-priming, DNA replication, 264897-1312-5998.1promoter activity, genome integration, or episomal concatermerization. The 5’ and 3’ elements are generally located at or near the 5’ and 3’ terminal end of the recombinant viral vector nucleic acid and can be naturally occurring or modified versions of naturally occurring sequences.Examples of 5’ and 3’ elements include adenovirus ITRs, adeno-associated virus ITRs, and packaging sequences; and retrovirus 5’ and 3’ long terminal repeats (LTRs) and packaging sequences. (Naso et aL, (2017) BioDaigs, 31(4), 317-334; Bulcha et L, (2021 ) Sig. Transduct. Target Ther. 6:53 (2021); and Liu and Seol (2020) BMB Reports; 53(11) :565 -575.)

[0125] The term “recombinant,” as a modifier of a vector indicates a combination of elements that does not occur in nature. For example, a recombinant viral vector nucleic acid provides 5’ and / or 3’ viral elements along with an expression cassette containing one or more elements not naturally associated with the 5’ and / or 3’ elements. Similarly, a viral vector, such as an rAAV vector may contain a naturally occurring or modified capsid, encapsidating recombinant viral vector nucleic acid.

[0126] In certain embodiments, the viral vector nucleic acid sequence comprises a 5’ LTR and a 3’ LTR, or a 5’ ITR and a 3’ ITR and (1) an expression cassette as described in Section IV. A. supra; and / or (2) a transgene encoding a protein, biologically active nucleic acid, or gene editing nucleic acid or protein as provided in Sections IV.A., IV.B., or IV. C. supra.

[0127] In certain embodiments, the viral vector further comprises a polyA signal operably linked to the 3’ ITR, where the polyA signal antagonizes potential transcription initiating from the 3’- ITR. The operably linked polyA signal is upstream of the 3 ’-ITR.

[0128] In certain embodiments, the viral vector nucleic acid contains any of 0-5, 0-10, 0-15, 0- 50, 0-100, or 0-150 CpGs; 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 CpGs; 0%, about 0.5%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% CpGs; and / or up to about 0.5%, up to about 1.0%, up to about 2.0%, up to about 3.0%, up to about 4.0%, up to about 5.0%, up to about 6%, up to about 7%, up to about 8%, up to about 9%, up to about 10%, up to about 11%, up to about 12%, up to about 13%, up to about 14% or up to about 15% CpGs.

[0129] 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 evoking 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-reactivity274897-1312-5998.1differences 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).

[0130] 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

[0131] 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., (202.1) Sig. Transduct. Target Ther. 6:53.)

[0132] 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. In an embodiment, the 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. Preferred 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 ah, PNAS (2000) 97(3): 1002-1007, each of which are hereby incorporated by reference herein in their entirety.)

[0133] 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.)

[0134] 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. (Liu284897-1312-5998.1and Seol (2020) BMB Reports, 53(11) : 565-575 ; and Bulcha et al., (2021 ) Sig. Transduct. TargetTher. 6:53.)V.B. Recombinant AAV Vectors

[0135] 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.

[0136] A rAAV vector contains AAV recombinant nucleic acid and a viral capsid. The rAAV recombinant nucleic acid lacks one or more AAV proteins 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.

[0137] 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, stuff er 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, about 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.

[0138] 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 294897-1312-5998.1can 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.)

[0139] 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.

[0140] 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.

[0141] 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 AAVl / rh.10; or VP1 of SEQ ID NO: 67 or SEQ ID NO: 70.

[0142] In certain embodiments, an AAV capsid 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 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, AAVl / rh.10, SEQ ID NO: 67 or SEQ ID NO: 70; 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.)

[0143] Recombinant AAV capsid comprising VP1 of SEQ ID NO: 67 is described, for example, in U.S. Patent No. 9,840,719; and rAAV capsids comprising VP1 of SEQ ID NO: 70 is described, for example, in U.S. Patent No. 9,169,299; both of these patents are incorporated herein by reference.304897-1312-5998.1

[0144] In certain embodiments, the capsid comprises VP1 having the sequence of SEQ ID NO: 67; VP2 having the sequence of SEQ ID NO: 68; and VP3 having the sequence of SEQ ID NO: 69.

[0145] 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 AAVl / rh.10), U.S. Patent No. 9,585,971, and Goertsen et al., (2022) Nat. Neurosci. 25, 106-115 (2022), each of which are incorporated by reference herein in its entirety.

[0146] 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, hereby incorporated by reference herein in its entirety.)

[0147] 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 HBoVl 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, both of which are incorporated by reference herein in their entirety.)

[0148] 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 a viral (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.

[0149] 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.314897-1312-5998.1

[0150] Recombinant AAV 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., (2017) Biotechnol. J. 12(3), 1600193; and International Application No. PCT / US2017 / 024951; the disclosures of which are herein incorporated in its entirety.

[0151] Recombinant AAV can be cultured under a variety of different conditions suitable for providing cell growth and gene expression. References describing rAAV manufacturing include Clement 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.)

[0152] 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 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 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 No. 9,408,904; and International Application Nos.PCT / US2017 / 025396 and PCT / US2016 / 064414, the disclosures of which are herein incorporated in their entirety.

[0153] 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.

[0154] In certain embodiments, a rAAV vector is produced by culturing a rAAV permissive cell comprising an AAV genome plasmid, where the rAAV permissive cell further 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 rep324897-1312-5998.1gene; (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.

[0155] In certain embodiments involving the use of HSV helper functions, the helper functions are provided by genes encoding for at least UL5, UL8, UL52, and ICP8.

[0156] In certain embodiments involving the use of adenovirus helper functions, helper functions are provided by genes encoding for at least E1A, E1B19K, E1B55K, E2A, E4orf6 and VA RNA. In certain embodiments El, E2A and VR RNA functions are provided by a helper plasmid, where additional helper functions are provided by a host strain.

[0157] In certain embodiments, rAAV vector is obtained by producing rAAV using methods described herein and purifying the rAAV. Purification of rAAV can performed using techniques such as gradient-based purification, column-based, and combined methods. (See, e.g., Ayuso et al., (2010), Curr Gene Ther. 10(6): 423 -36, hereby incorporated by reference herein in its entirety.)V.C. Retrovirus Vectors

[0158] 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 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. Non- Viral Vectors

[0159] In certain embodiments, a delivery vehicle is a non-viral vector. Non-viral vectors include nanoparticles and naked nucleic acid. Preferred non-viral vectors are nanoparticles. A variety of different nanoparticles can be employed including lipid nanoparticles (LNP), polymeric nanoparticles, lipid polymer nanoparticles (LPNP), protein and peptide-based nanoparticles, DNA dendrimers and DNA-based nanocarriers, carbon nanotubes, microparticles, microcapsules, inorganic nanoparticles, peptide cage nanoparticles, and exosomes. (See, e.g., Riley and Vermerris Nanomaterials (2017) 201, 7, 94; Thomas et al., Molecules (2019), 24, 3744; Bochicchio et al., (2021), 13, 198; Munagala et al., Cancer Letters (2021), 505, 58; Fu et 334897-1312-5998.1al., (2020) NanoImpact 20, 100261; Neshat et al., (2020) Current Opin. Biotechnol. 66: 1-10; Ouranidis et al., (2022) Biomedicines, 10, 50; and Qin et al., Signal Transduct Target Ther. (2022) May 21 ;7( 1 ): 166, each of which are hereby incorporated by reference herein in their entirety.) Non-viral vectors are further described, for example, in International Patent Publication No. WO2023196188, hereby incorporated by reference herein in its entirety.VII. Host Immune Response

[0160] In some cases, vector administration can result in an undesirable immune response due to, for example, vector components, the transgene product being recognized as foreign, or an edited gene producing a protein being regarded as foreign. In such cases, if desired, the host immune response can be reduced, for example, using an immune cell modulator or immunosuppressant. In some cases, such as cancer treatment, viral treatment and bacterial treatment certain responses may be advantageous.

[0161] In certain embodiments a phagocyte-depleting agent is used to reduce a subject immune response. Methods, compounds and compositions for depletion of phagocytic immune cells are described, for example, by International Patent Publication No. WO2022140788A1 (hereby incorporated by reference herein in its entirety).

[0162] In certain embodiments a cGAS-STING pathway and / or inflammasome pathway inhibitor is used to reduce a subject immune response. Methods, compounds and compositions for inhibiting the cGAS-STING pathway and / or the inflammasome pathway are described, for example, by International Publication Patent No. W02023004437, hereby incorporated by reference herein in its entirety.

[0163] In certain embodiments, administration is in combination with an immunosuppressive agent or regimen. Such agents and regimens can be utilized, as needed, to achieve immune tolerance or mitigate the immune response to the produced protein, the provided polynucleotides, or the provided delivery vehicles. Examples of immunosuppressive agents and regimens include methotrexate, rituximab, intravenous gamma globulin (IVIG), omalizumab, ImmTOR® (synthetic vaccine particle (SVP)-rapamycin (rapamycin encapsulated in a biodegradable nanoparticle)), ImmTOR-IL™ (ImmTOR with Treg-selective IL-2 agonist), B- cell depletion, immunoadsorption, and plasmapheresis.

[0164] In certain embodiments, the viral vector or non-viral vector is administered in conjunction with one or more immunosuppressive agents, where one or more immunosuppressive agent is administered prior to, substantially at the same time as, or after, administering the vector or non-viral vector. In certain embodiments, the one or more immunosuppressive agent is administered concomitantly with the vector or non-viral vector. In344897-1312-5998.1certain embodiments, the one or more immunosuppressive agents is administered 1-12, 12-24 or 24-48 hours; or 2-4, 4-6, 6-8, 8-10, 10-14, 14-20, 20-25, 25-30, 30-50 days, or more than 50 days prior to viral or non-viral vector administration. In certain embodiments, the one or more immunosuppressive agent is administered 1-12, 12-24 or 24-48 hours; or 2-4, 4-6, 6-8, 8-10, 10- 14, 14-20, 20-25, 25-30, 30-50 days, or more than 50 days, following viral or non-viral vector administration. Administration of immunosuppressive agents after a period of time following administering vector or non-viral vector can be done, for example, if there is a decrease in the encoded protein after the initial expression levels for a period of time, e.g., 20-25, 25-30, 30-50, 50-75, 75-100, 100-150, 150-200 or more than 200 days following vector or non-viral vector administration.

[0165] In certain embodiments, the immunosuppressive agent is an anti-inflammatory agent. In certain embodiments, the immunosuppressive agent is a steroid, e.g., a corticosteroid. In certain embodiments, the immunosuppressive agent is prednisone, prednisolone, calcineurin inhibitor (e.g., cyclosporine, tacrolimus), MMF (mycophenolic acid, e.g. CellCept®, Myfortic®), CD52 inhibitor (e.g., alemtuzumab), CTLA4-Ig (e.g., abatacept, belatacept), anti-CD3 mAb, anti-LFA- 1 mAb (e.g., efalizumab), anti-CD40 mAb (e.g., ASKP1240), anti-CD22 mAb (e.g., epratuzumab), anti-CD20 mAb (e.g., rituximab, orelizumab, ofatumumab, veltuzumab), proteasome inhibitor (e.g., bortezomib), TACI-Ig (e.g., atacicept), anti-C5 mAb (e.g., eculizumab), mycophenolate, azathioprine, sirolimus everolimus, TNFR-Ig, anti-TNF mAb, tofacitinib, anti-IL-2R (e.g., basiliximab), anti-IL-17 mAb (e.g., secukinumab), anti-IL-6 mAb (e.g., anti-IL-6 antibody sirukumab, anti-IL-6 receptor antibody tocilizumab (Actemra®), IL- 10 inhibitor, TGF-beta inhibitor, a B cell targeting antibody (e.g., rituximab), a mammalian target of rapamycin (mTOR) inhibitor (e.g., rapamycin), synthetic vaccine particle (SVP™)-rapamycin (rapamycin encapsulated in a biodegradable nanoparticle), intravenous gamma globulin (IVIG), omalizumab, methotrexate, a tyrosine kinase inhibitor (e.g., ibrutinib), cyclophosphamide, fmgolimod, an inhibitor of B-cell activating factor (BAFF) (e.g., anti-BAFF mAb, e.g., belimumab), an inhibitor of a proliferation-inducing ligand (APRIL), anti-IL-lb mAb (e.g., canakinumab (Haris®)), a C3a inhibitor, a Tregitope (see, e.g., U.S. Patent No. 10,213,496), or a combination and / or derivative thereof.

[0166] Immune-suppression protocols, including the use of rapamycin, alone or in combination with IL- 10, can be used to decrease, reduce, inhibit, prevent or block humoral and cellular immune responses to protein. In certain embodiments, hepatic gene transfer with viral vector (e.g., rAAV) and non-viral vector can be used to induce immune tolerance to protein through induction of regulatory T cells (Tregs).354897-1312-5998.1VIII, Pharmaceutical Compositions

[0167] Pharmaceutical compositions comprise a pharmaceutical acceptable carrier facilitating administration and / or storage of the fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain, encoding mRNA, viral vectors or non-viral vectors. Reference to “pharmaceutically acceptable” indicates the components do not cause substantial undesirable biological effects at the amount utilized. Pharmaceutically 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 No. WO2021 / 071835.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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 or364897-1312-5998.1immunosuppressive agents), treatments, protocols, or therapeutic regimens; and provide for a long or short term response.

[0172] Pharmaceutical compositions comprising transgenes can be delivered to a subject, to allow production of the encoded protein or biologically active nucleic acid. Delivery can be in vivo or ex vivo. In certain embodiments, pharmaceutical compositions comprise sufficient genetic material to enable a recipient to produce a therapeutically effective amount of a protein or biologically active nucleic acid in the subject.

[0173] 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 takes into account observed symptoms and / or biomarkers associated with a particular disease or disorder. Selection of a particular effective dose can be optimized 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.

[0174] In certain embodiments, a pharmaceutical composition comprising a rAAV vector comprises empty AAV capsids. In certain embodiments, in a pharmaceutical composition comprising rAAV vectors and empty AAV capsids, the ratio of empty AAV capsids to rAAV vector is within or between about 100:1-50: 1, from about 50: 1-25: 1, from about 25: 1-10: 1, from about 10: 1-1 : 1, from about 1 : 1-1 : 10, from about 1 : 10-1 :25, from about 1 :25-1 :50, or from about 1 : 50- 1 : 100. In certain embodiments, the ratio of the empty AAV capsids to the rAAV vector is about 2:1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, or 10: 1.

[0175] 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.IX, Administration and Treatment

[0176] A fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain or encoding mRNA can be used, for example, as a therapeutic for the treatment of diseases mediated by an IgG immune response, to facilitate 374897-1312-5998.1treatment that may evoke IgG immune response, and for research purposes. Examples of treatment that may evoke a pathologic or otherwise undesirable IgG response include tissue and organ transplant, autoimmunity, and the use of gene delivery vehicles, such as viral vectors.

[0177] In certain embodiments the fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain or encoding mRNA 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); antineutrophil cytoplasmic antibody-associated vasculitides (ANCA associated vasculitis); Wegener granulomatosis; Churg-Strauss syndrome; microscopic polyangiitis; anti-NMDAR Encephalitis; anti-phospholipid antibody syndrome (APS) and catastrophic APS; autoimmune bullous skin diseases, Pemphigus, Pemphigus foliaceus (PF), fogo selvagem (FS) (endemic form); pemphigus vulgaris (P V); 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 (IIP); 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 (REED); rheumatic fever; rheumatoid arthritis (RA); serum-sickness, immune complex hypersensitivity (type III); Sjogren Syndrome (SS); SEE 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.)

[0178] In certain embodiments, the fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain or encoding mRNA is administered to reduce an immune response to organ or tissue transplant, such as kidney transplant or liver transplant.

[0179] In certain embodiments, the fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain or encoding mRNA is384897-1312-5998.1administered 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.B. supra and Section 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.).

[0180] Reference to “treatment” or “treat” refers to both prophylactic, 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, suspected 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.

[0181] 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.

[0182] The terms “effective amount” and “sufficient amount” is that amount required to obtain a desired effect, such as being sufficient to degrade native IgG in a subject. In certain embodiments, the amount is sufficient to obtain a clinical meaningful amelioration in at least one symptom or cause associated with a disease or disorder.

[0183] When used as a therapeutic, in different embodiments, the amount is sufficient to cause a reduction in IgG of about 10% to about 25%, about 25% to about 50%, about 50% to about 75%, about 75% to about 80%, about 80% to 100%, or about 95% to about 99%. In further embodiments the reduction in IgG is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.

[0184] In certain embodiments when used to facilitate treatment that may evoke an Ig immune response, the amount is sufficient to cause a reduction in IgG of about 10% to about 25%, about 25% to about 50%, about 50% to about 75%, about 75% to about 80%, about 80% to 100%, or about 95% to about 99%. In further embodiments the reduction in IgG is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.394897-1312-5998.1

[0185] An effective amount can be administered alone or can be administered 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 and adverse side effects.

[0186] 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.

[0187] In certain embodiments, the fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain is administered at a dosage of about 0.01 mg / kg to about 10 mg / kg body mass of a subject. For example, a suitable dosage may be from about 0.05 mg / kg to about 5 mg / kg body mass of a subject, or from about 0.1 mg / kg to about 4 mg / kg body weight of a subject.

[0188] In certain embodiments, encoding mRNA is administered at a dosage of about 0.01 mg / kg to about 10 mg / kg body mass of a subject.

[0189] Preferred treatment doses of vectors for gene therapy can vary depending on the particular vector and different factors such as the type, onset, progression, severity, frequency, duration of a particular disease or disorder to which the treatment is directed; the clinical endpoint desired; previous or simultaneous treatments; and the general health, age, gender, race or immunological competency 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.

[0190] 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 level or protein 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.

[0191] In certain embodiments, reduction of expression or activity of a targeted protein is reduced. In different embodiments reduction of expression or activity of a protein targeted by a404897-1312-5998.1therapeutic 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.

[0192] 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. Pat. No. 5,720,720). Depending on the composition, for example, delivery can be subcutaneously, epidermally, intradermally, intrathecally, intraorbitally, intramucosally, intraperitoneally (IP), intravenously (IV), intrapleurally, intraarterially, orally, intrahepatically, via the portal vein, intracranial or intramuscularly.

[0193] In certain embodiments, the fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain or encoding mRNA 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, Cl 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), or a disease of solid organs (e.g., brain, liver, kidney, heart).

[0194] In certain embodiments, the fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain or encoding mRNA 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 a-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.414897-1312-5998.1

[0195] 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 by myoblasts 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.

[0196] In certain embodiments, the fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain or encoding mRNA 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 Cl deficiency or gamma-carboxylase deficiency.

[0197] 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.

[0198] 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 is 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 considered normal, due to functional clotting which can be determined, for example, by an activated partial thromboplastin time (aPTT) one-stage clotting assay.

[0199] In certain embodiments, the fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain or encoding mRNA is used in conjugation with gene therapy to treat bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC); over- anti coagulation 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.424897-1312-5998.1

[0200] In certain embodiments, the fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain or encoding mRNA 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, sicklecell anemia, sickle cell disease, Fanconi’s anemia, diabetes, amyotrophic lateral sclerosis (ALS), myotubularin myopathy, 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 disease 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.

[0201] In certain embodiments, the fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain or encoding mRNA 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, or Parkinson’s disease. In certain embodiments, the disease is a psychiatric disease, an addiction (e.g., to tobacco, alcohol, or drugs), 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).IX, A, Administration Timing with a Treatment Evoking Immune Response434897-1312-5998.1

[0202] In different embodiments administration of a fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain or encoding mRNA is at or about the same time as a treatment that can evoke an immune response, 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, (18) up to about 72 hours, (19) up to about 96 hours, or (20) up to about a week prior to the treatment that can evoke an immune response. In further embodiments, the fusion protein or encoding mRNA is administered within at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or at about the time indicated any of (1) to (20). For examples, administration ranges for (1) up to five minutes in different embodiments include about 3 to about 5 minutes (within about 60%), about 3.75 to about 5 minutes (within about 70%), about 4 to about 5 minutes (within about 80%), about 4.5 to about 5 minutes (within about 90%), or about 5 minutes.

[0203] In further embodiments, the fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain or encoding mRNA, is administered up to 72 hours, 1-72 hours, 6-12 hours, 12-48 hours, or 48-72 hours prior to a treatment that can evoke an immune response.

[0204] In different embodiments administration of a fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain or encoding mRNA is at or about the same time as viral vector gene therapy, 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, (18) up to about 72 hours, (19) up to about 96 hours, or (20) up to about a week prior to the gene therapy treatment. In further embodiments, the fusion protein or mRNA is administered within at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or at about the time indicated any of (1) to (20).

[0205] In further embodiments, the fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain or encoding mRNA is added up to 72 hours, 1-72 hours, 6-12 hours, 12-48 hours, or 48-72 hours prior to viral vector gene therapy.444897-1312-5998.1

[0206] In different embodiments administration of a fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain or encoding mRNA is at or about the same time as an organ or tissue transplant, 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, (18) up to about 72 hours, (19) up to about 96 hours, or (20) up to about a week prior to the organ or tissue transplant. In further embodiments, the fusion protein or mRNA is administered within at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or at about the time indicated any of (1) to (20).

[0207] In further embodiments, the fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain or encoding mRNA is added up to 72 hours, 1-72 hours, 6-12 hours, 12-48 hours, or 48-72 hours prior to organ or tissue transplant. IX.B, Administration Timing after an Adverse Immune Response

[0208] In different embodiments the fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain or encoding mRNA is administered after an adverse immune response to a treatment is observed; and / 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, (18) up to about 72 hours, (19) up to about 96 hours, or (20) up to about a week after an adverse IgG immune response is observed. In further embodiments, the fusion protein or mRNA is administered within at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or at about the times indicated in any of (1) to (20).IX.C. Subsequent Administration

[0209] In certain embodiments, a subject is administered a second viral vector and a fusion protein comprising an immunoglobulin G-degrading enzyme domain or encoding mRNA. The timing for the administration of the second viral vector and fusion protein or encoding mRNA can be the same as described above for the viral vector and fusion protein or mRNA. In further embodiments, the second viral vector is the same as the first viral vector and the subject is being re-dosed. Re-dosing can occur, for example, years or decades after the initial dose.454897-1312-5998.1X, Kits

[0210] Further provided herein is a kit comprising in separate containers: (a) a pharmaceutical composition comprising a fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain or encoding mRNA; 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 hereinXI, Additional Aspects and Embodiments

[0211] Additional aspects, embodiments, and examples of combinations thereof include the following:

[0212] A first aspect is directed to a fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain.

[0213] El further describes the first aspect, wherein said immunoglobulin G-degrading enzyme domain comprises an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to any of SEQ ID NOs: 2 and 25-61, or comprising any of SEQ ID NOs: 2 and 25-61. In a further embodiment, said immunoglobulin G-degrading enzyme comprises an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2 or comprising SEQ ID NO: 2.

[0214] E2 further describes El, wherein said human serum albumin binding domain comprises an antibody variable region that binds human serum albumin.

[0215] E3 further describes E2, wherein said antibody variable region is an IgNAR variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 65 and a CDR3 comprising the amino acid of SEQ ID NO: 66.

[0216] E4 further describes E2, wherein said antibody variable region comprises three CDRs wherein a first CDR comprises the amino acid sequence of SEQ ID NO: 62, a second CDR comprises the amino acid sequence of SEQ ID NO: 63, and a third CDR comprises the amino acid sequence of SEQ ID NO: 64.

[0217] E5 further describes the first aspect and El, wherein said human serum albumin binding domain comprises an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 3, 4, 5 or 14, or comprises any of SEQ ID NOs: 3, 4, 5 or 14. In a further embodiment, said human serum albumin binding domain comprises an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 14, or comprises SEQ ID NO: 14.464897-1312-5998.1

[0218] E6 further describes each of the first aspect, El, E2, E3, E4, and E5, wherein said immunoglobulin G-degrading enzyme domain comprises an amino acid sequence of SEQ ID NO: 2 or differing from SEQ ID NO: 2 by 1 to 10 amino acids.

[0219] E7 further describes each of the first aspect, El, E2, E3, E4, E5, and E6, wherein said fusion protein comprises the configuration (amino to carboxy): ABS-L-IDE, wherein ABS is said human serum albumin binding domain, L is an optionally present first amino acid linker, and IDE is said immunoglobulin G-degrading enzyme domain.

[0220] E8 further describes E7, wherein said first linker is present and comprises at least one amino acid. In further embodiments, said linker comprises at least 2, 3, 4, 5, 10, 20, 30, 40, 50, 60 or 70 amino acids.

[0221] E9 further describes E7, wherein said first linker is not present. In such a case, the fusion protein comprises the configuration (amino to carboxy): ABS-IDE.

[0222] E10 further describes each of the first aspect, El, E2, E3, E4, E5, and E6, wherein said fusion comprises the configuration (amino to carboxy): IDE-L-ABS, wherein ABS is said human serum albumin binding domain, L is an optional present first amino acid linker, and IDE is said immunoglobulin G-degrading enzyme domain.

[0223] El 1 further describes E10, wherein said first linker is present and comprises at least one amino acid. In further embodiments, said linker comprises at least 2, 3, 4, 5, 10, 20, 30, 40, 50, 60 or 70 amino acids.

[0224] E12 further describes El l, wherein said first linker is not present. In such a case, said fusion protein comprises the configuration (amino to carboxy) IDE-ABS.

[0225] E13 further describes E8 or El l, wherein said first linker comprises up to 50 amino acids and comprises an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the sequence of any of SEQ ID NOs: 8-12, or comprises the sequence of any of SEQ ID NOs: 8-12.

[0226] E14 further describes each of the first aspect, El, E2, E3, E4, E5, E6, E7, E8, E9, E10, El l, E12, and E13, wherein said fusion protein comprises an amino acid sequence at least 95%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 15-24, comprises the sequence of any of SEQ ID NOs: 15-24, or comprises a modified version of any of SEQ ID NOs: 15-24, lacking the N-terminal methionine. In a further embodiment said fusion protein comprises an amino acid sequence at least 97% identical, at least 98%, or at least 99% to any of SEQ ID NOs: 17, 18 or 19, comprises SEQ ID NOs: 17, 18 or 19 or comprises a modified version of SEQ ID NOs: 17, 18 or 19 lacking the N-terminal methionine.

[0227] El 5 further describes the first aspect, wherein said fusion protein comprises the sequence of any of SEQ ID NOs: 15-19 or a derivative thereof lacking the N-terminal methionine present 474897-1312-5998.1in said sequence. In a further embodiment, said fusion protein comprises the sequence of SEQ ID NO: 18 or a derivative thereof lacking the N-terminal methionine present in said sequence; or comprises the sequence of SEQ ID NO: 19 or a derivative thereof lacking the N-terminal methionine present in said sequence.

[0228] E16 further describes said fusion protein of each of the first aspect, El, E2, E3, E4, E5, E6, E7, E8, E9, E10, El l, E12, E13, E14 and E15 further comprising a purification tag joined to either the N-terminus or C-terminus of said fusion protein through a protease cleavage site and / or a second amino acid linker.

[0229] A second aspect is directed to a polynucleotide comprising a sequence encoding said fusion protein of any of the first aspect, El, E2, E3, E4, E5, E6, E7, E8, E9, E10, El l, E12, E13, E14, E15 and E16.

[0230] El 7 further describes the second aspect, wherein the sequence encoding said fusion protein is operably linked to a promoter.

[0231] E18 further describes E17, wherein said polynucleotide is an expression cassette. In a further embodiment, said polynucleotide is a DNA expression vector.

[0232] E19 further describes the second aspect, E17 or E18, wherein said polynucleotide is an mRNA construct further comprising a 5’-cap, a 5’UTR, a 3’UTR, and a poly(A) tail.

[0233] A third aspect is directed to a recombinant cell comprising said polynucleotide of any one of the second aspect, El 7, E18 or E19.

[0234] E20 further describes the third aspect, wherein said recombinant cell is a prokaryotic cell. In a further embodiment the prokaryotic cell is E. coli.

[0235] A fourth aspect is directed to a method of producing said fusion protein, comprising culturing said recombinant cell of the second aspect or E20. In a further embodiment, said fusion protein is purified.

[0236] A fifth aspect is directed to a pharmaceutical composition comprising said fusion protein of any of the first aspect, El, E2, E3, E4, E5, E6, E7, E8, E9, E10, El l, E12, E13, E14, E15, E16, E17, or E18, or the mRNA of E19, and pharmaceutically acceptable carrier.

[0237] A sixth aspect is directed to a method of decreasing IgG in a subject comprising administering to said subject said fusion protein of any of the first aspect, El, E2, E3, E4, E5, E6, E7, E8, E9, E10, El l, E12, E13, E14, E15, E16, E17, or E18; or the mRNA of E19; or the pharmaceutically composition of the fifth aspect.

[0238] E21 further describes the sixth aspect, wherein said method is used treat an autoimmune disease or immune-mediated condition in said subject.484897-1312-5998.1

[0239] E22 further describes the sixth aspect, wherein said method is used in conjunction with an organ or tissue transplant. In a further embodiment, said transplant is kidney or liver transplant.

[0240] E23 further describes the sixth aspect, E21 and E22 wherein the timing said fusion protein administration is as provided in Section IX. A. or Section IX.B supra. In a further embodiment said fusion protein or encoding mRNA is administered up to 72 hours, 1-72 hours, 6-12 hours, 12-48 hours, or 48-72 hours prior to an organ or tissue transplant, such as a kidney or liver transplant.

[0241] E24 further describes each of the sixth aspect, E21, E22, and E23, wherein said fusion protein or said pharmaceutical composition comprising said fusion protein is administered.

[0242] E25 further describes each of the sixth aspect, E21, E22, E23, and E24, wherein said subject is a human.

[0243] A seventh aspect is directed to a method of administering a transgene to a subject comprising the steps of administering to said subject (a) a first viral vector comprising said transgene; and (b) said fusion protein of any of the first aspect, El, E2, E3, E4, E5, E6, E7, E8, E9, E10, E11, E12, E13, E14, E15, E16, E17, and E18; said mRNA of El 9; or said pharmaceutically composition of the fifth aspect.

[0244] E26 further describes the seventh aspect wherein the timing of the viral vector and said fusion protein administration is as provided in Section IX. A. supra. In a further embodiment said fusion protein or encoding mRNA is administered up to 72 hours, 1-72 hours, 6-12 hours, 12-48 hours, or 48-72 hours prior to said viral vector gene therapy.

[0245] E27 further describes the seventh aspect and E26, wherein said method further comprises administration of a second viral vector, wherein said second viral vector may be the same or different than said first viral vector; and an immunoglobulin G-degrading enzyme is added prior to said second viral vector. In a further embodiment, said immunoglobulin G-degrading enzyme is a fusion protein described herein.

[0246] E28 further describes E27, wherein second viral vector comprises either (1) the same capsid as said first viral vector; or (2) a capsid that evokes immunologically cross-reactive antibodies with said first viral vector capsid.

[0247] E29 further describes the seventh aspect, E26, E27 and E28, wherein said first viral vector is a recombinant adeno-associated virus (rAAV).

[0248] E30 further describes the seventh aspect, E26, E27, E28 and E29, wherein said subject is a human.

[0249] E31 further describes the seventh aspect, E26, E27, E28, E29, and E30, wherein said transgene encodes a viral antigen, a bacterial antigen, a therapeutic protein, a short hair pin RNA 494897-1312-5998.1(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), or a transcription activator-like effector nuclease (TALEN).XII, Sequences

[0250] Table 2 provides different nucleic acid and amino acid sequences. In certain embodiments, a polynucleotide comprises a nucleic acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or identical to any of the nucleic acid sequences provided in Table 2. In certain embodiments, a polypeptide comprises a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or identical to any of the amino acid sequences provided in Table 2.Table 2504897-1312-5998.1514897-1312-5998.1524897-1312-5998.1534897-1312-5998.1544897-1312-5998.1554897-1312-5998.1564897-1312-5998.1EXAMPLES

[0251] 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.

[0252] Example 1 : IgG Cleavage in Pooled Human Plasma

[0253] Recombinant protein IdeS was produced in E. coli. The native leader sequence of IdeS (AB045752.1) was removed and replaced with a sequence encoding an 8x-Histidine tag. This modified IdeS gene was synthesized and cloned into an expression plasmid and then transformed into bacteria. Recombinant His-tagged IdeS protein was purified via nickel immobilized metal affinity chromatography (Ni-IMAC) and anion exchange chromatography574897-1312-5998.1(AEX). Yield and concentration were determined by A280; purity was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and reversed-phase high- performance liquid chromatography (RP-HPLC); aggregation was measured by size exclusion chromatography (SEC); and endotoxin levels were determined by limulus amebocyte lysate (LAL) assay.

[0254] IdeS (WT) and IdeS variants (Table 3) were added to human pooled normal plasma and incubated at 37 °C for one hour. IdeS (1.5 pL volume) was added to pooled normal plasma (18.5 pL volume) for an estimated ratio of 1 gram of IdeS per 9,000 grams of human IgG. The input quantity of IdeS-PK-Seq variants was normalized to relative molecular weight to achieve molar equivalency of IdeSTgG between test articles. Digested samples were diluted 1 : 16,000 in 0. IX sample buffer (Protein Simple) for a non-reducing 60-440 kDa Wes™ capillary electrophoresis assay. Digest samples were run in duplicate Wes™ capillaries. Detection was performed using a goat anti-human IgG F(ab’)2 antibody (Jackson #109-005-006, lot 138190) and a Wes™ antigoat secondary detection module (ProteinSimple, Cat. No. DM-006).

[0255] Table 3

[0256] FIG. 1 illustrates the Wes™ peak area of each F(ab’ ^-containing IgG-fragment as a percent of total Wes™ peak area in each sample. The Wes™ peak areas of intact IgG, singlecleaved IgG fragment, and F(ab’)2 fragment were determined using Compass software (ProteinSimple). The peak area of each band was calculated as a percentage of total peak area (intact + single-cleaved + F(ab’)2). Samples shown in FIG. 1 were performed in duplicate.

[0257] Example 2: Albumin Binding

[0258] The ability of different albumin-binding IdeS variants to bind to native albumin from multiple species was evaluated using an ELISA. ELISA plates (96 well) were coated with 100 pL, 0.1 pg / well Human Serum Albumin (HSA) (Sigma, Cat: A3782), Cynomolgus macaque Serum Albumin (CSA) (MyBioSource, Cat: MBS135765), Rabbit Serum Albumin (RSA), or Mouse Serum Albumin (MSA) (Innovative Research, Cat: IMSALB). Plates were blocked with 300 pL / well of Superblock T20. IdeS and IdeS-Seq variants were serially diluted in blocking buffer and added to the plates 100 pL / well for one hour at room temperature. Detection was performed with 1 : 10,000 goat anti-FabRICATOR® (anti-IdeS) antibody that was commercially584897-1312-5998.1sourced (Genovis, Cat. No. A0-FR1) and affinity purified. Secondary antibody anti-goat IgG conjugated to HRP (Jackson Labs, Cat no 305-035-046) was diluted to 1 : 10k and added at 100 pL / well. One step TMB Ultra was added at 100 pL / well. Color development was quenched with the addition of 100 pL 2N sulfuric Acid. Absorbance at OD 450 was read on a Biotek Synergy Hl plate reader.

[0259] The tested variants are summarized in Table 3. IdeS refers to wild-type IdeS (SEQ ID NO: 2). The results are shown in FIGs. 2A-2F.

[0260] Example 3: Serum Half-Life of Albumin Binding Variants

[0261] Recombinant IdeS and IdeS variants (Table 3) were administered to dual -transgenic hFcRn+ / +, hAlb+ / + mice that express human neonatal Fc-receptor (FcRn) and human serum albumin under the control of their respective endogenous mouse promoters. Each animal received a single intravenous injection of IdeS or IdeS-variant protein. Pre-formulated test article (0.5 mg / mL total protein in Dulbecco’s phosphate buffered saline) was administered to each animal via intravenous tail vein injection (200 pL injection volume), resulting in an approximate dose between 1.5 mg / kg and 4.2 mg / kg of IdeS variant protein. Whole blood was collected serially via saphenous vein micro-sampling into serum tubes. Blood was allowed to clot for 30 minutes to 2 hours at room temperature prior to centrifugation at 10,000 x g at 23 °C for 5 minutes. Serum samples were frozen at -80 °C.

[0262] The presence and relative concentration of IdeS or IdeS variants in serum samples over time was determined by Wes™. Serum samples were thawed on ice. Each Wes™ experiment consisted of serially-collected test article and included 4 capillaries of quality controls (QCs). Sera from extra untreated mice were pooled to form a negative QC; this pooled sera was run in triplicate on each Wes™ plate. IdeS or IdeS variants were spiked into pooled sera of extra untreated mice at a concentration of at 12 pg / mL to generate a positive QC; each positive QC was run once on the relevant Wes™ plate. Test articles and QCs were diluted 1 : 100 in 0. lx Sample Buffer (ProteinSimple). Diluted samples (4 pL) were combined with 1 pL of 5x fluorescent master mix containing dithiothreitol (DTT; ProteinSimple), followed by incubation at 95 °C for 5 minutes. Goat anti-FabRICATOR® (anti-IdeS) primary antibody (Genovis, Cat. No. A0-FR1) was diluted 1 :250 in milk-free antibody diluent (ProteinSimple). Wes™ 12-230 kDa protein assay (ProteinSimple 25-Capillary Separation Module, Cat. No. SM-W004) setup followed manufacturer’s instructions using a goat secondary horseradish peroxidase (HRP)- conjugated detection reagent (ProteinSimple Anti -Goat Detection Module, Cat. No. DM-006). The IdeS peak in serum was identified using Compass for Simple Western (SW) software version 4.0.0 (ProteinSimple).594897-1312-5998.1

[0263] Table 4 summarizes the half-life of different variants. The different variants are further described in Table 3. The relative half-life of each IdeS variant in plasma was determined by serial blood collections and semi-quantitative capillary electrophoretic Wes™ detection of IdeS in serum with an anti-IdeS antibody (anti-FabRICATOR®). Estimated group mean [range] halflife was determined from a 1 -phase decay best fit of IdeS variant Wes™ IdeS-peak height versus elapsed time in individual animals. Significance (p < 0.05) of mean IdeS-PK variant half-life relative to IdeS half-life was determined by one-way ANOVA with Dunnett’s test for multiple comparisons.

[0264] Table 4

[0265] The results illustrated in Table 4 indicate that albumin-binding IdeS variants IdeS-PK-8 and IdeS-PK-9 demonstrate significantly (p < 0.05) extended half-life relative to IdeS in a transgenic mouse model of human serum albumin and human FcRn interaction. Other albuminbinding IdeS-PK variants also show a trend toward increased mean half-life relative to IdeS.

[0266] FIGs. 3 A-3E illustrate one-phase decay of albumin-binding IdeS variants in sera of hFcRn+ / +, hAlb+ / + transgenic mice. Each animal (GN-XXX) received a single intravenous dose of IdeS or IdeS-variant protein. The relative decay and half-life of each IdeS variant in plasma was determined by serial blood collections and quantitative capillary electrophoretic Wes™ detection of IdeS in serum with an anti-IdeS antibody (anti-FabRICATOR®). Data points indicate IdeS or IdeS-PK-Seq variant Wes™ histogram peak heights as a function of time. A best-fit 1 -phase decay curve was fitted to data points for each individual animal. The dotted horizontal line in each panel represents the peak height mean + 3 standard deviations of 3 technical replicates of pooled sera from untreated animals (i.e., negative QC, respective IdeS variant Wes™ assay “noise”). FIG. 3 A illustrates results with native IdeS (SEQ ID NO: 2), FIG. 3B illustrates results with the construct of SEQ ID NO: 20, FIG. 3C illustrates results with the construct of SEQ ID NO: 21, FIG. 3D illustrates results the with construct of SEQ ID NO: 22, FIG. 3E illustrates results with the construct of SEQ ID NO: 23, and FIG. 3F illustrates results the construct of SEQ ID NO: 24. Albumin-binding IdeS variants demonstrate distinct serum604897-1312-5998.1pharmacokinetics relative to IdeS in a transgenic mouse model of human serum albumin and human FcRn interaction.

[0267] The IdeS peak height was used to quantify the relative amount of IdeS within each group over time. A nonlinear 1 -phase decay least-squares fit of IdeS peak height versus elapsed time post-dose (hours) was performed in GraphPad Prism 8.4.3 to estimate the approximate in vivo half-life. For each animal, Wes™ peak heights were also normalized as a percentage of peak height at Timepoint 1 (FIG. 4A and FIG. 4B).

[0268] FIGs. 4A and 4B illustrate estimated half-life of albumin-binding IdeS variants (see Table 3) and decline in hFcRn+ / +, hAlb+ / + transgenic mouse sera over time. Recombinant IdeS and IdeS variant (IdeS-PK-Seq) proteins were administered to dual -transgenic hFcRn+ / +, hAlb+ / + mice expressing human neonatal Fc-receptor (FcRn) and human serum albumin under the control of their respective endogenous mouse promoters. Each animal received a single intravenous dose of IdeS or IdeS-variant protein. The relative decay and half-life of each IdeS variant in plasma was determined by serial blood collections and quantitative capillary electrophoretic Wes™ detection of IdeS in serum with an anti-IdeS antibody (anti- FabRICATOR®). Albumin-binding IdeS variants demonstrate extended half-life and slower decay relative to IdeS in a transgenic mouse model of human serum albumin and human FcRn interaction.

[0269] FIG. 4A provides a best-fit 1-phase decay curve of IdeS peak height versus elapsed time used to determine the half-life of IdeS (WT) or albumin-binding IdeS variant (Seq) in each study subject. Data points represent the half-life determined for each animal, and bars indicate the mean ± range half-life of n = 3 - 4 animals per group. A one-way ANOVA with Dunnett’s test for multiple comparisons was performed to determine statistical significance of mean half-life for each IdeS variant (Seq) relative to IdeS (WT) (*, p < 0.05, ****, p < 0.0001).

[0270] FIG. 4B provides, for each animal, Wes™ peak height at each timepoint normalized as a percentage of peak height at the first serum collection (approximately 30-minutes postinjection). Mean ± SD normalized peak height per group was plotted for each serum collection timepoint (horizontal line, 100%).

[0271] 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.614897-1312-5998.1

Claims

CLAIMSI / we claim:

1. A fusion protein comprising an immunoglobulin G-degrading enzyme domain and a human serum albumin binding domain.

2. The fusion protein of claim 1, wherein said immunoglobulin G-degrading enzyme comprises an amino acid sequence at least 90% identical to any of SEQ ID NOs: 2 and 25-613. The fusion protein of claim 2, wherein said human serum albumin binding domain comprises an antibody variable region that binds human serum albumin.

4. The fusion protein of claim 3, wherein said antibody variable region is an IgNAR variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 65 and a CDR3 comprising the amino acid of SEQ ID NO: 66.

5. The fusion protein of claim 3, wherein said antibody variable region comprises three CDRs wherein a first CDR comprises the amino acid sequence of SEQ ID NO: 62, a second CDR comprises the amino acid sequence of SEQ ID NO: 63, and a third CDR comprises the amino acid sequence of SEQ ID NO: 64.

6. The fusion protein of claim 1, wherein said human serum albumin binding domain comprises an amino acid sequence at least 90% identical to any of SEQ ID NOs: 3, 4, 5 or 14.

7. The fusion protein of claim 6, wherein said human serum albumin binding domain comprises the amino acid sequence of SEQ ID NO: 14.

8. The fusion protein of any one of claims 1-7, wherein said immunoglobulin G-degrading enzyme domain comprises an amino acid sequence of SEQ ID NO: 2 or differing from SEQ ID NO: 2 by 1 to 10 amino acids.

9. The fusion protein of claim 8, wherein said immunoglobulin G-degrading enzyme domain comprises the amino acid sequence of SEQ ID NO: 2.

10. The fusion protein of any of claims 1-9, wherein said fusion protein comprises the configuration (amino to carboxy): ABS-L-IDE, wherein ABS is said human serum albumin binding domain, L is an optionally present first amino acid linker, and IDE is said immunoglobulin G-degrading enzyme domain.

11. The fusion protein of claim 10, wherein the first linker is present and comprises at least one amino acid.

12. The fusion protein of 10, wherein said fusion protein comprises the configuration (amino to carboxy): ABS-IDE.

13. The fusion protein of any one of claims 1-9, wherein said fusion protein comprises the configuration (amino to carboxy): IDE-L-ABS,624897-1312-5998.1wherein ABS is said human serum albumin binding domain, L is an optionally present first amino acid linker, and IDE is said immunoglobulin G-degrading enzyme domain.

14. The fusion protein of claim 13, wherein L is present and comprises at least one amino acid.

15. The fusion protein of claim 13, wherein said fusion protein comprises the configuration (amino to carboxy): IDE-ABS.

16. The fusion protein of claims 11 or 14, wherein said first linker comprises up to 50 amino acids and comprises an amino acid sequence at least 90% identical to the sequence of any of SEQ ID NOs: 8-12.

17. The fusion protein any one of claims 1-16, wherein the fusion protein comprises an amino acid sequence at least 95% identical to any of SEQ ID NOs: 15-24.

18. The fusion protein of claim 17, wherein the fusion protein comprises an amino acid sequence at least 97% identical to SEQ ID NOs: 18 or 19.

19. The fusion protein of claim 1, comprising the sequence of any of SEQ ID NOs: 15-19 or a derivative thereof lacking the N-terminal methionine present in said sequence.

20. The fusion protein of claim 19, comprising the sequence of SEQ ID NO: 18 or a derivative thereof lacking the N-terminal methionine present in said sequence.

21. The fusion protein of any one of claims 1-20, further comprising a purification tag joined to either the N-terminus or C-terminus of the fusion protein through a protease cleavage site and / or a second amino acid linker.

22. A polynucleotide comprising a sequence encoding the fusion protein of any one of claims 1-21.

23. The polynucleotide of claim 22, wherein the sequence encoding the fusion protein is operably linked to a promoter.

24. The polynucleotide of claim 23, wherein said polynucleotide is an expression cassette.

25. The polynucleotide of claim 23, wherein said polynucleotide in a DNA expression vector.

26. An mRNA construct comprising the polynucleotide of claim 22, wherein the polynucleotide is an RNA polynucleotide and the construct comprises: a 5’-cap, a 5’UTR, the RNA polynucleotide, a 3’UTR, and a poly(A) tail.

27. A recombinant cell comprising the polynucleotide of any one of claims 22-26.

28. The recombinant cell of claim 27, wherein said recombinant cell is a prokaryotic cell.

29. The recombinant cell of claim 28, wherein said prokaryotic cell is E. coli.634897-1312-5998.

130. A method of producing said fusion protein comprising culturing the recombinant cell of any one of claims 27-29.

31. A pharmaceutical composition comprising the fusion protein of any one of claims 1-21 or the mRNA construct of claim 26, and a pharmaceutically acceptable carrier.

32. The pharmaceutical composition of claim 31, wherein said composition comprises said fusion protein.

33. A method of decreasing IgG in a subject comprising administering to said subject, said fusion protein of any one of claims 1-21, the mRNA construct of claim 26, or the pharmaceutically composition of claims 31 or 32.

34. The method of claim 33, wherein the method is used to treat an autoimmune disease or immune-mediated condition in said subject.

35. The method of claim 33, wherein said method is used in conjunction with a liver or organ transplant.

36. The method of claim 35, wherein said transplant is a kidney or liver transplant.

37. The method of any one of claims 33-36, wherein said fusion protein or said pharmaceutical composition comprising said fusion protein is administered.

38. The method of any one of claims 33-37, wherein said subject is a human.

39. A method of administering a transgene to a subject comprising the steps of administering to the subject (a) the fusion protein of any one of claims 1-21, the mRNA construct of claim 26, or the pharmaceutical composition of claims 31 or 32; and (b) a first viral vector comprising the transgene.

40. The method of claim 39, wherein said step (b) comprises said fusion protein or a pharmaceutical composition comprising said fusion protein.

41. The method of claims 39 or 40, wherein said step (b) is performed after said step (a).

42. The method of any one of claims 39-41, wherein said step (a) is performed up to 72 hours prior to said step (a).

43. The method of any one of claims 39-41, wherein said step (a) is performed 6-12 hours prior to said step (a).

44. The method of claim any one of claims 39-43, further comprises administration of a second viral vector, wherein said second viral vector may be the same or different than said first viral vector; and an immunoglobulin G-degrading enzyme is added prior to the second viral vector.

45. The method of claim 44, wherein the second viral vector comprises either (1) the same capsid as the first viral vector; or (2) a capsid that evokes immunologically cross-reactive antibodies with the first capsid.644897-1312-5998.

146. The method of any one of claims 39-45, wherein said first viral vector is a recombinant adeno-associated virus (rAAV).

47. The method of any one of claims 39-46, wherein said subject is a human.

48. The method of any of one of claims 39-47, wherein said 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), or a transcription activator-like effector nuclease (TALEN).654897-1312-5998.1

Images