Compositions and methods for modulating expression of therapeutic transgenes

Abstract

Featured are compositions and methods of use in conjunction with gene therapy agents. In particular, the disclosure features compositions and methods designed to permanently and / or site- specifically alter expression of a gene therapy agent comprising a therapeutic transgene.

Description

[0001] PATENT

[0002] ATTORNEY DOCKET NO.: 51772-016WO2

[0003] COMPOSITIONS AND METHODS FOR MODULATING EXPRESSION OF THERAPEUTIC TRANSGENES

[0004] SEQUENCE LISTING

[0005] This application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on September 29, 2025, is named “51772-016WO2_Sequence_Listing_9_29_25. xml” and is 99,822 bytes in size.

[0006] TECHNICAL FIELD

[0007] The present disclosure relates to methods of modulating the expression of a heterologous gene of interest, as well as vectors, nucleic acids, and related compositions that may be used in such methods.

[0008] BACKGROUND

[0009] Recombinant vectors derived from adeno-associated viruses (AAVs), among other gene therapy modalities, have become a prevalent paradigm for heterologous gene expression due, at least in part, to the remarkable safety profile of this non-pathogenic virus, as well as its potential to achieve transgene expression in a variety of tissues. Intramuscularly administered AAV vectors have been explored as a vehicle for the delivery of various transgenes, including therapeutic and prophylactic proteins. Following the delivery of a gene therapy agent, however, there are instances in which it may be beneficial - or even necessary - to suppress transgene expression, such as for individuals who require an additional therapeutic intervention that is contraindicated for the originally delivered gene therapy modality. Limited options are available for terminating expression of a transgene following delivery with an AAV vector or other durable gene therapy vehicle. Accordingly, there remains a need for compositions and methods that can be used to attenuate transgene expression following the delivery of a gene therapy agent.

[0010] SUMMARY OF THE INVENTION

[0011] The present disclosure relates to compositions and methods for modulating heterologous gene expression. For example, the disclosure provides modalities for achieving recombinase-mediated attenuation of the expression of a gene that has been previously provided to a subject by way of a gene therapy agent, such as a recombinant adeno-associated virus (AAV) or another durable gene therapy vector, such as another gene therapy vector described herein.

[0012] The compositions and methods of the disclosure address important safety risks that may arise after administering a gene therapy agent to a subject. In certain scenarios, it may be desirable to attenuate the expression of a previously provided gene of interest. This need can originate from any of various causes, such as the gene therapy agent being administered in a manner or quantity that results in transgene expression beyond therapeutic levels or safely tolerated levels. Additionally or alternatively, following administration of the gene therapy agent, the subject's condition may change in a manner that renders the previously administered gene therapy agent presently contraindicated for the subject.

[0013] The compositions and methods described herein provide solutions to these potential problems by attenuating the expression of the previously provided transgene. This can be achieved through non- PATENT

[0014] ATTORNEY DOCKET NO.: 51772-016WO2 specific reduction of expression of the gene of interest throughout the subject’s body or by selectively targeting specific cell types, tissues, organs, or organ systems in which reducing gene expression is desired. Specifically, to achieve this reduction in gene expression, the gene therapy agent may be designed so as to contain recombinase recognition sites flanking the gene of interest or components of the gene therapy agent that facilitate gene expression. Following administration of the gene therapy agent, excision of the gene of interest - and a consequent reduction in gene expression - can be triggered by administering a recombinase that specifically binds and cleaves or rearranges DNA intervening between these recognition sites.

[0015] The sections that follow describe non-limiting examples of the gene therapy agents, recombinase enzymes, and recombinase recognition sites that may be used in conjunction with the compositions and methods of the disclosure, as well as non-limiting examples of the types of subjects that may be treated using this paradigm.

[0016] In one aspect, the disclosure features a method of reducing expression or activity of a protein of interest in a subject (e.g., a human subject) in need thereof, the method comprising:

[0017] (a) administering to the subject a recombinant nucleic acid comprising (i) a transgene expression cassette comprising a transgene encoding the protein of interest and (ii) a pair of recombinase recognition sites flanking the 5’ and 3’ ends of the expression cassette or any functional component thereof; and

[0018] (b) subsequently administering to the subject a corresponding recombinase that specifically binds the recombinase recognition sites, thereby inactivating the transgene.

[0019] In another aspect, the disclosure features a method of reducing expression or activity of a protein of interest in a subject in need thereof, the method comprising administering to the subject a recombinase, wherein the subject has previously been administered a recombinant nucleic acid comprising (i) a transgene expression cassette comprising a transgene encoding the protein of interest and (ii) a pair of recombinase recognition sites flanking the 5’ and 3’ ends of the expression cassette or any functional component thereof, wherein the recombinase administered to the subject specifically binds the recombinase recognition sites, thereby inactivating the transgene. In some embodiments, the subject is a mammal (e.g., a human).

[0020] In a further aspect, the disclosure features a method of eliminating expression or activity of a protein of interest in an undesired tissue or organ of a subject while preserving expression or activity of the protein of interest in a desired tissue or organ of the subject, the method comprising:

[0021] (a) administering to the subject a recombinant nucleic acid comprising (i) a transgene expression cassette comprising a transgene encoding the protein of interest and (ii) a pair of recombinase recognition sites flanking the 5’ and 3’ ends of the expression cassette or any functional component thereof, wherein the recombinant nucleic acid is administered to the subject systemically and / or wherein upon administration, the recombinant nucleic acid biodistributes to a plurality of tissues or organs; and

[0022] (b) subsequently administering to the subject a corresponding recombinase that specifically binds the recombinase recognition sites, wherein the recombinase is specifically administered to and / or specifically biodistributes to the undesired tissue or organ, and is neither specifically administered to nor specifically biodistributes to the desired tissue or organ, thereby inactivating the transgene in the PATENT

[0023] ATTORNEY DOCKET NO.: 51772-016WO2 undesired tissue or organ while preserving the transgene in the desired tissue or organ. In some embodiments, the subject is a mammal (e.g., a human).

[0024] In yet another aspect, the disclosure features a method of reducing expression or activity of a protein of interest in an undesired tissue or organ of a subject while preserving expression or activity of the protein of interest in a desired tissue or organ of the subject, the method comprising administering to the subject a recombinase, wherein the subject has previously been administered a recombinant nucleic acid comprising (i) a transgene expression cassette comprising a transgene encoding the protein of interest and (ii) a pair of recombinase recognition sites flanking the 5’ and 3’ ends of the expression cassette or any functional component thereof. In some embodiments, the recombinant nucleic acid has been administered to the subject systemically. In some embodiments, upon administration, the recombinant nucleic acid biodistributes to a plurality of tissues or organs. In some embodiments, the recombinase administered to the subject specifically binds the recombinase recognition sites. In some embodiments, the recombinase is specifically administered to and / or specifically biodistributes to the undesired tissue or organ, and is neither specifically administered to nor specifically biodistributes to the desired tissue or organ, thereby inactivating the transgene in the undesired tissue or organ while preserving the transgene in the desired tissue or organ. In some embodiments, the subject is a mammal (e.g., a human).

[0025] In a further aspect, the disclosure features a method of reducing expression or activity of a protein of interest in a subject from a level that exceeds a therapeutic window for that protein in the subject to a level that is within a safe and effective range for the subject. The method may include:

[0026] (a) administering to the subject a recombinant nucleic acid comprising (i) a transgene expression cassette comprising a transgene encoding the protein of interest and (ii) a pair of recombinase recognition sites flanking the 5’ and 3’ ends of the expression cassette or any functional component thereof;

[0027] (b) measuring the level of expression or activity of the protein of interest in the subject and, if it is determined that the level of expression or activity is higher than that which is safely tolerated by the subject,

[0028] (c) administering to the subject a corresponding recombinase (e.g., in a sub-saturating amount) that specifically binds the recombinase recognition sites, thereby inactivating the transgene. In some embodiments, the subject is a mammal (e.g., a human).

[0029] In another aspect, the disclosure features a method of reducing expression or activity of a protein of interest in a subject from a level that exceeds a therapeutic window for that protein in the subject to a level that is within a safe and effective range for the subject. The method may include:

[0030] (a) administering to the subject a recombinant nucleic acid comprising (i) a transgene expression cassette comprising a transgene encoding the protein of interest and (ii) a pair of recombinase recognition sites flanking the 5’ and 3’ ends of the expression cassette or any functional component thereof;

[0031] (b) determining that the level of expression or activity of the protein of interest in the subject is higher than that which is safely tolerated by the subject; and PATENT

[0032] ATTORNEY DOCKET NO.: 51772-016WO2

[0033] (c) administering to the subject a corresponding recombinase (e.g., in a sub-saturating amount) that specifically binds the recombinase recognition sites, thereby inactivating the transgene. In some embodiments, the subject is a mammal (e.g., a human).

[0034] In still another aspect, the disclosure features a method of reducing expression or activity of a protein of interest in a subject from a level that exceeds a therapeutic window for that protein in the subject to a level that is within a safe and effective range for the subject, the method comprising administering to the subject a recombinase (e.g., in a sub-saturating amount). In some embodiments, the subject has previously been administered a recombinant nucleic acid comprising (i) a transgene expression cassette comprising a transgene encoding the protein of interest and (ii) a pair of recombinase recognition sites flanking the 5’ and 3’ ends of the expression cassette or any functional component thereof. In some embodiments, following administration of the recombinant nucleic acid to the subject, the subject has been determined to exhibit a level of expression or activity of the protein of interest that is higher than that which is safely tolerated by the subject. In some embodiments, the recombinase administered to the subject specifically binds the recombinase recognition sites, thereby inactivating the transgene. In some embodiments, the subject is a mammal (e.g., a human).

[0035] In some embodiments, the pair of recombinase recognition sites flank the 5’ and 3’ ends of the expression cassette. In some embodiments, the pair of recombinase recognition sites flank the 5’ and 3’ ends of the transgene. In some embodiments, the pair of recombinase recognition sites flank the 5’ and 3’ ends of any functional component of the expression cassette. In some embodiments, the functional component is a promoter. In some embodiments, the functional component is a muscle-specific promoter. In some embodiments, the muscle-specific promoter is a phosphoglycerate kinase (PGK) promoter, a desmin promoter, a muscle creatine kinase promoter, a myosin light chain promoter, a myosin heavy chain promoter, a cardiac troponin C promoter, a troponin I promoter, a myoD gene family promoter, an actin alpha promoter, an actin beta promoter, an actin gamma promoter, or a promoter within intron 1 of ocular paired like homeodomain 3, a cytomegalovirus promoter, or a chicken-p-actin promoter. In some embodiments, the functional component is an enhancer. In some embodiments, the enhancer is a muscle-specific enhancer. In some embodiments, the muscle-specific enhancer is a muscle creatine kinase enhancer, a distal regulatory element (DRE) enhancer, a smooth muscle myosin heavy chain (SMHC) enhancer, a dystrophin intron 1 enhancer, a MyoD enhancer, or a myocyte enhancer factor 2 (MEF2) enhancer. In some embodiments, the functional component is a terminator, and further, the terminator is in a reverse orientation relative to the transgene. In some embodiments, the terminator is a polyadenylation signal (pA).

[0036] In some embodiments, the recombinant nucleic acid is administered to the subject in the form of a recombinant viral vector comprising the nucleic acid. In some embodiments, the recombinant viral vector is a recombinant adeno-associated viral (AAV) vector. In some embodiments, the recombinant AAV vector is a serotype 1 , 2, 3, 4, 5, 6, 7, 8, 9, rh10, or rh74 AAV vector. In some embodiments, the AAV vector is encapsulated by one or more synthetic capsid proteins.

[0037] In some embodiments, expression of the transgene that encodes the protein of interest is sustained following administration of the recombinant nucleic acid to the subject. In some embodiments, expression of the transgene that encodes the protein of interest is sustained for between one week and PATENT

[0038] ATTORNEY DOCKET NO.: 51772-016WO2 ten years following administration, between 1 week and 7 years, between 1 week and 5 years, between 1 week and 3 years, between 1 week and 1 year, between 2 weeks and 24 weeks, or between 4 weeks and 6 weeks following administration. In some embodiments, expression is sustained for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks, at least 16 weeks, at least 17 weeks, at least 18 weeks, at least 19 weeks, at least 20 weeks, at least 21 weeks, at least 22 weeks, at least 23 weeks, at least 24 weeks, at least 25 weeks, or at least 26 weeks following administration.

[0039] In some embodiments, expression of the transgene that encodes the protein of interest is sustained within fifty percent of an initial expression level. In some embodiments, expression of the transgene that encodes the protein of interest is sustained within twenty-five percent of an initial expression level. In some embodiments, expression of the transgene that encodes the protein of interest is sustained within twenty percent of an initial expression level. In some embodiments, expression of the transgene that encodes a protein of interest is sustained within fifteen percent of an initial expression level. In some embodiments, expression of the transgene that encodes a protein of interest is sustained within ten percent of an initial expression level. In some embodiments, expression of the transgene that encodes a protein of interest is sustained within nine percent, eight percent, seven percent, six percent, five percent, four percent, three percent, two percent, or one percent of an initial expression level.

[0040] In some embodiments, the recombinase is administered to the subject in the form of a nucleic acid encoding the recombinase. In some embodiments, the recombinase is administered to the subject by way of a viral vector comprising the nucleic acid encoding the recombinase. In some embodiments, the viral vector comprising the nucleic acid encoding the recombinase is an AAV vector. In some embodiments, the AAV vector is a serotype 1 , 2, 3, 4, 5, 6, 7, 8, 9, rh10, or rh74 AAV vector, or the AAV vector is encapsulated by one or more synthetic capsid proteins.

[0041] In some embodiments, the recombinase is administered to the subject in the form of a lipid nanoparticle (LNP) comprising (i) the recombinase or (ii) a nucleic acid encoding the recombinase, such as an RNA (e.g., mRNA) or DNA polynucleotide encoding the recombinase.

[0042] In some embodiments, the recombinase is administered to the subject in a tissue-specific or organ-specific manner. In some embodiments, the recombinase is administered to directly to the subject’s muscle tissue or liver.

[0043] In some embodiments, the recombinase is a Cre recombinase and the recombinase recognition sites are lox sites.

[0044] In some embodiments, the Cre recombinase has an amino acid sequence that is at least 85% identical (e.g., at least 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of any one of SEQ ID NOs: 17-19.

[0045] In some embodiments, the lox sites comprise a pair of lox sites set forth in Table 2, herein.

[0046] In some embodiments, administration of the recombinase reduces expression of the protein of interest by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, as compared to expression of the protein of interest in the subject prior to administration of the recombinase. PATENT

[0047] ATTORNEY DOCKET NO.: 51772-016WO2

[0048] In some embodiments, administration of the recombinase completely eliminates transgene expression, such that expression of the transgene is no longer detectable. In some embodiments, administration of the recombinase reduces the expression of the protein of interest, e.g., by inactivating the transgene in a subset of cells in a cell type, tissue, tissue system, organ, or organ system of interest.

[0049] In some embodiments, administration of the recombinase reduces the expression of the protein of interest by about 2-fold, by about 3-fold, by about 4-fold, by about 5-fold, by about 6-fold, by about 7- fold, by about 8-fold, by about 9-fold, by about 10-fold, by about 15-fold, by about 20-fold, by about 25- fold, by about 30-fold, by about 35-fold, by about 40-fold, by about 45-fold, by about 50-fold, or greater than 50-fold, as compared to expression of the protein of interest in the subject prior to administration of the recombinase. In some embodiments, administration of the recombinase reduces the expression of the protein of interest by about 2-fold, by about 3-fold, by about 4-fold, by about 5-fold, by about 6-fold, by about 7-fold, by about 8-fold, by about 9-fold, by about 10-fold, by about 11 -fold, by about 12-fold, by about 13-fold, by about 14-fold, by about 15-fold, by about 16-fold, by about 17-fold, by about 18-fold, by about 19-fold, by about 20-fold, by about 21 -fold, by about 22-fold, by about 23-fold, by about 24-fold, by about 25-fold, by about 26-fold, by about 27-fold, by about 28-fold, by about 29-fold, by about 30-fold, by about 31 -fold, by about 32-fold, by about 33-fold, by about 34-fold, by about 35-fold, by about 36-fold, by about 37-fold, by about 38-fold, by about 39-fold, by about 40-fold, by about 41 -fold, by about 42-fold, by about 43-fold, by about 44-fold, by about 45-fold, by about 46-fold, by about 47-fold, by about 48-fold, by about 49-fold, by about 50-fold, or by greater than 50-fold, as compared to expression of the protein of interest in the subject prior to administration of the recombinase.

[0050] In some embodiments, the recombinase is administered to the subject at least one week, one month, or one year following administration of the recombinant nucleic acid comprising the transgene encoding the protein of interest. In some embodiments, the recombinase is administered at least one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, or 24 weeks following administration of the recombinant nucleic acid comprising the transgene encoding the protein of interest, optionally wherein the recombinase is administered between one week and ten years following administration of the recombinant nucleic acid comprising the transgene encoding the protein of interest.

[0051] In some embodiments, the recombinase is administered to the subject from one month to one year following administration of the recombinant nucleic acid comprising the transgene encoding the protein of interest.

[0052] In some embodiments, the recombinase is administered to the subject from one year to ten years following administration of the recombinant nucleic acid comprising the transgene encoding the protein of interest.

[0053] In some embodiments, the recombinase is administered to the subject in advance of the subject commencing treatment with a therapeutic intervention that is contraindicated with the protein of interest, optionally wherein the therapeutic intervention is to be chronically administered to the subject. PATENT

[0054] ATTORNEY DOCKET NO.: 51772-016WO2

[0055] In some embodiments, the subject has had an immunogenic response against the protein of interest. In some embodiments, the protein of interest accumulated to levels that have induced toxicity in the subject.

[0056] In some embodiments, the therapeutic intervention is an antiviral, antibacterial, or other microbial agent. In some embodiments, the therapeutic intervention is a chemotherapy and / or immunotherapy regimen. In some embodiments, the therapeutic intervention is one or more immunosuppressive agents.

[0057] In some embodiments, the recombinase is administered to the subject from one day to 12 months prior to the subject receiving a therapeutic intervention that is contraindicated with the protein of interest, optionally wherein the therapeutic intervention is to be chronically administered to the subject.

[0058] In some embodiments, the recombinase is administered to the subject one week, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, 11 months, or 12 months prior to receiving a therapeutic intervention that is contraindicated with the protein of interest, optionally wherein the therapeutic intervention is to be chronically administered to the subject.

[0059] In some embodiments, the recombinase is administered to the subject in a single dose. In some embodiments, the recombinase is administered to the subject in a plurality of doses. In some embodiments, each dose is separated by at least one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, or ten weeks.

[0060] BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The accompanying drawings are included to illustrate embodiments of the disclosure and further an understanding of its implementations.

[0062] FIG. 1 is a graph showing serum protein concentrations (pM) of GLP-1 over time following intramuscular administration of an AAV (adeno-associated viral) vector containing a GLP-1 transgene. FIG. 1 abbreviations: GLP-1 : Glucagon-like peptide-1 ; pM: picomolar.

[0063] FIG. 2A shows the design of the vector construct, AAV-GLP-1 -STOP, in which the transgene GLP-1 is flanked by the loxP sites. The vector construct is further described in Example 2, below. FIG. 2A abbreviations: GLP-1 : Glucagon-like peptide-1 ; cmv: cytomegalovirus; pA: polyadenylation signal site.

[0064] FIG. 2B is a graph showing the amount of secreted GLP-1 for 2-4 days post lipid nanoparticle (LNP) delivery, in accordance with the procedure described in Example 2, below. The graph presents the results obtained in connection with both negative control mRNA and Cre mRNA. The experiment was performed in vitro in 293 cells and the amount of GLP-1 production from the AAV-GLP-1 -STOP construct was measured using the protocol outlined in Example 2. The amount of secreted GLP-1 (pM) was plotted against the day post LNP delivery. FIG. 2B abbreviations: LNP: lipid nanoparticle; GLP-1 : Glucagon-like peptide-1 .

[0065] FIG. 3A shows the design of the vector construct AAV-GLP-1 -STOP, in which the transgene GLP-1 is flanked by loxP sites, and the vector construct AAV-GLP-1 , which does not contain loxP sites. The vector construct is further described in Examples 6 and 7, below. FIG. 3A abbreviations: GLP-1 : Glucagon-like peptide-1 ; cmv: cytomegalovirus; pA: polyadenylation signal site. PATENT

[0066] ATTORNEY DOCKET NO.: 51772-016WO2

[0067] FIG. 3B is a graph showing the amount of serum GLP-1 (pM) at 14 days post-AAV administration, in accordance with the procedure described in Example 6, below. The graph presents the results obtained from administration of AAV-GLP-1 -STOP and AAV-GLP-1 vectors.

[0068] FIG. 3C is a graph showing the amount of serum GLP-1 at 28 days post-AAV administration, in accordance with the procedure described in Example 6, below. The graph presents the results of mice who were administered either AAV-GLP-1 -STOP or AAV-GLP-1 and dosed with either PBS or LNP-Cre at 14 days post-AAV administration. Serum GLP-1 expression is plotted as a fraction of serum GLP-1 expression on day 14.

[0069] FIG. 3D is a graph showing the amount of serum GLP-1 over 24 weeks post-AAV administration, in accordance with the procedure described in Example 6, below. The graph presents the results of mice who were administered AAV-GLP-1 -STOP at day 0 and treated with PBS or LNP-Cre at days 14, 70, 1 12, and 147. The amount of serum GLP-1 is shown relative to the PBS-treated group and is plotted relative to the study day.

[0070] FIG. 4 is a graph showing the amount of serum GLP-1 at 60 days post-AAV administration, in accordance with the procedure described in Example 7, below. These experiments were performed in mice, who were given either PBS or varying doses of LNP-Cre at 46 days post-AAV administration. The amount of serum GLP-1 is shown as a fraction of serum GLP-1 on day 46.

[0071] FIG. 5A shows the design of the vector constructs AAV-GLP-1 -STOP, AAV-GLP-1 -STOP2, and AAV-GLP-1 -STOP3, in which the transgene GLP-1 is flanked by alternate loxP sites. The vector construct is further described in Example 8, below.

[0072] FIG. 5B is a graph showing the amount of serum GLP-1 expression at 42 days post-AAV administration, in accordance with the procedure described in Example 8, below. These experiments were performed in mice, who were given either PBS or LNP-Cre at 28 days post-AAV administration. The amount of serum GLP-1 is shown as a fraction of serum GLP-1 on day 28.

[0073] FIG. 6A shows possible outcomes of the action of Cre recombinase on DNA containing loxP sites in various orientations.

[0074] FIG. 6B shows the design of the vector constructs ‘control’ and ‘PA’ constructs 1 through 10, in which the pA site following the transgene GLP-1 is flanked by alternate lox sites in various orientations. The vector constructs are further described in Example 9, below.

[0075] FIG. 6C shows the experimental timeline of AAV and LNP administrations and blood draws. The experiment is further described in Example 9, below.

[0076] FIG. 6D is a graph showing the amount of serum GLP-1 expression at 42 days post-AAV administration, in accordance with the procedure described in Example 9, below. These experiments were performed in mice, who were given one of the AAV constructs described in Example 9, below, on day 0, followed by LNP-Cre at 30 days post-AAV administration. The amount of serum GLP-1 is shown as a fraction of serum GLP-1 on day 30. PATENT

[0077] ATTORNEY DOCKET NO.: 51772-016WO2

[0078] DEFINITIONS

[0079] Unless otherwise defined herein, scientific, and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of "or" means "and / or" unless stated otherwise. The use of the term "including," as well as other forms, such as "includes" and "included," is not limiting.

[0080] As used herein, the term "about," as applied to one or more values of interest, refers to a value that falls within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of a stated reference value, unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0081] As used herein, the term “comprise” or variations thereof (e.g., comprises or comprising) will be understood to include a feature or a plurality of features but not exclude additional features or plurality of features.

[0082] As used herein in the context of a therapeutic intervention and a protein of interest, the term “contraindicated” refers to instances in which administration of the therapeutic intervention (e.g., a small molecule, biologic drug product, cell therapy, gene therapy, surgical procedure, dietary supplement, or other therapeutic agent intended to produce a beneficial effect in a patient) would either negate the effect of, or produce toxicity as a result of, expression of the protein of interest. Exemplary contraindications that may be encountered by a patient undergoing treatment with the compositions and methods of the disclosure are described herein. In such instances, a patient may be administered Cre recombinase to reduce or terminate expression of the protein of interest.

[0083] As used herein, the terms “polynucleotide” or “nucleic acid” refer to polymers of nucleotides of any length and include DNA and / or RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction. Thus, for instance, polynucleotides as defined herein include, without limitation, single- and double-stranded DNA, DNA including single- and double-stranded regions, single- and double-stranded RNA, and RNA including single- and doublestranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or include single- and double-stranded regions. In addition, the term “polynucleotide” as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. The term “polynucleotide” specifically includes complementary DNA (cDNA).

[0084] As used herein, the term “recombinant nucleic acid” refers to a nucleic acid that has been manipulated in a fashion that generally does not occur in nature. A recombinant nucleic acid may be formed by molecular biology methods that combine genetic material from two or more different biological sources, thereby creating nucleic acid sequences that are not naturally occurring.

[0085] A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their PATENT

[0086] ATTORNEY DOCKET NO.: 51772-016WO2 analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after synthesis, such as by conjugation with a label. Other types of modifications include, for example, “caps,” substitution of one or more of the naturally-occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, and the like) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, and the like), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, and the like), those with intercalators (e.g., acridine, psoralen, and the like), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, and the like), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid or semi-solid supports. The 5’ and 3’ terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2’-Omethyl-, 2’-O-al lyl-, 2’-fluoro-, or 2’-azido-ribose, carbocyclic sugar analogs, a-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs, and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), “(O)NR2 (“amidate”), P(O)R, P(O)OR’, CO or CH2 (“formacetal”), in which each R or R’ is independently H or substituted or unsubstituted alkyl (1 -20 C) optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0087] As used herein, the terms “adenine” and “adenosine” are interchangeable terms in reference to a nucleotide that has an adenine base. As used herein, the terms “cytosine” and “cytidine” are interchangeable terms in reference to a nucleotide that has a cytosine base. As used herein, the terms “guanine” and “guanidine” are interchangeable terms in reference to a nucleotide that has a guanine base. As used herein, the terms “thymine” and “thymidine” are interchangeable terms in reference to a nucleotide that has a thymine base. As used herein, the terms “uracil” and “uridine” are interchangeable terms in reference to a nucleotide that has a uracil base.

[0088] As used herein, the term "nucleoside" refers to a molecule made up of a heterocyclic base and its sugar.

[0089] As used herein, the term "nucleotide" refers to a nucleoside having a phosphate group, or a variant thereof, on its 3' or 5' sugar hydroxyl group. Examples of phosphate group variants include, but are not limited to, saturated alkyl phosphonates, unsaturated alkenyl phosphonates, phosphorothioates, and phosphoramidites. PATENT

[0090] ATTORNEY DOCKET NO.: 51772-016WO2

[0091] In the context of this disclosure, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring (e.g., modified) portions that function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.

[0092] As used herein, a “coding sequence” refers to an open reading frame (ORF) in a nucleic acid that, upon expression, yields a polypeptide or protein. An ORF is a continuous stretch of DNA or RNA beginning with a start codon (e.g., methionine (ATG or AUG)) and ending with a stop codon (e.g., TAA, TAG or TGA, or UAA, UAG or UGA). An ORF typically encodes a protein. It will be understood that the sequences disclosed herein may further include additional elements, e.g., 5' and 3' UTRs, but that those elements, unlike the ORF, need not necessarily be present in an RNA polynucleotide (e.g., an mRNA transcript) disclosed herein.

[0093] As used herein, “messenger RNA,” “mRNA,” or “mRNA transcript” is any RNA molecule that encodes a (at least one) protein (e.g., a polypeptide) or fragment thereof and can be translated to produce the encoded protein or the fragment thereof in vitro, in vivo, in situ, or ex vivo. Structural and topological features as well as post-transcriptional modifications of mRNA are described herein and are well-known in the art.

[0094] As used herein, the terms “complementary,” “complementarity,” or variations thereof (e.g., “having complementarity” or “has complementarity”) refer to two nucleotides that form canonical Watson- Crick base pairs. For the avoidance of doubt, Watson-Crick base pairs in the context of the present disclosure include adenine-thymine, adenine-uracil, and cytosine-guanine base pairs. A proper Watson- Crick base pair is referred to in this context as a “match,” while each unpaired nucleotide, and each incorrectly paired nucleotide, is referred to as a “mismatch.” Alignment for purposes of determining percent nucleic acid sequence complementarity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software.

[0095] As used herein, the term “percent (%) sequence complementarity” with respect to a reference polynucleotide sequence is defined as the percentage of nucleic acids in a candidate sequence that are complementary to the nucleic acids in the reference polynucleotide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence complementarity. A given nucleotide is considered to be “complementary” to a reference nucleotide as described herein if the two nucleotides form canonical Watson-Crick base pairs. For the avoidance of doubt, Watson-Crick base pairs in the context of the present disclosure include adenine-thymine, adenine-uracil, and cytosine-guanine base pairs. A proper Watson-Crick base pair is referred to in this context as a “match,” while each unpaired nucleotide, and each incorrectly paired nucleotide, is referred to as a “mismatch.” Alignment for purposes of determining percent nucleic acid sequence complementarity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. PATENT

[0096] ATTORNEY DOCKET NO.: 51772-016WO2

[0097] Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal complementarity over the full length of the sequences being compared. As an illustration, the percent sequence complementarity of a given nucleic acid sequence, A, to a given nucleic acid sequence, B, (which can alternatively be phrased as a given nucleic acid sequence, A that has a certain percent complementarity to a given nucleic acid sequence, B) is calculated as follows:

[0098] 100 multiplied by (the fraction X / Y) where X is the number of complementary base pairs in an alignment (e.g., as executed by computer software, such as BLAST) in that program’s alignment of A and B, and where Y is the total number of nucleic acids in B. It will be appreciated that where the length of nucleic acid sequence A is not equal to the length of nucleic acid sequence B, the percent sequence complementarity of A to B will not equal the percent sequence complementarity of B to A. As used herein, a query nucleic acid sequence is considered to be “completely complementary” to a reference nucleic acid sequence if the query nucleic acid sequence has 100% sequence complementarity to the reference nucleic acid sequence.

[0099] As used herein, “peptide” is less than or equal to 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0100] As used herein, “polypeptide” refers to a polymer of amino acid residues linked together by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. Typically, however, a polypeptide will be at least 50 amino acids and the polypeptide is termed a peptide. If the polypeptide is a peptide, it will be about 5 amino acid residues long. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the foregoing. A polypeptide may be a single molecule or may be a multi-molecular complex such as a dimer, a trimer, or a tetramer. The term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analog of a corresponding naturally occurring amino acid.

[0101] As used herein, the terms “protein” refers to contiguous amino acids or amino acid residues. Typically, proteins have a function. However, proteins also encompass polypeptides and smaller contiguous amino acid sequences that do not have a functional activity. The functional proteins of this invention include, but are not limited to, enzymes, dehydrogenases, hydrolases, oxidoreductases, transferases, lyases, ligases, receptors, receptor ligands, cytokines, antibodies, immunomodulatory molecules, signaling molecules, or proteins that are tagged or modified (e.g., for diagnostic or other clinical applications). Useful general classes of enzymes include, but are not limited to, proteases, cellulases, lipases, hemicellulases, laccases, amylases, glucoamylases, esterases, lactases, polygalacturonases, galactosidases, ligninases, oxidases, peroxidases, glucose isomerases, nitrilases, hydroxylases, polymerases and depolymerases. In addition to enzymes, the encoded proteins which can be used in this invention include, but are not limited to, transcription factors, antibodies, receptors, growth factors (any of the PDGFs, EGFs, FGFs, SCF, HGF, TGFs, TNFs, insulin, IGFs, LIFs, oncostatins, and CSFs), immunomodulators, peptide hormones, cytokines, integrins, interleukins, adhesion molecules, thrombomodulatory molecules, protease inhibitors, angiostatins, defensins, cluster of differentiation antigens, interferons, chemokines, antigens including those from infectious viruses and organisms, PATENT

[0102] ATTORNEY DOCKET NO.: 51772-016WO2 oncogene products, thrombopoietin, erythropoietin, tissue plasminogen activator, and any other biologically active protein which is desired for use in a clinical setting. Such proteins are well known in the art. Also included are deletion mutants of such proteins (e.g., in which an individual residue or a plurality of residues are deleted from the sequence), individual domains of such proteins, fusion proteins made from such proteins, and mixtures of such proteins.

[0103] As used herein, the term “antibody” refers to a molecule that specifically binds to, or is immunologically reactive with, a particular antigen and includes at least the variable domain of a heavy chain, and normally includes at least the variable domains of a heavy chain and of a light chain of an immunoglobulin. Antibodies and antigen-binding fragments, variants, or derivatives thereof include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-ld) antibodies. Antibody molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG 1 , lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass of immunoglobulin molecule. Moreover, unless otherwise indicated, the term “monoclonal antibody” (mAb) is meant to include both intact molecules as well as antibody fragments (such as, for example, Fab and F(ab')2 fragments) that are capable of specifically binding to a target protein. Fab and F(ab')2 fragments lack the Fc fragment of an intact antibody.

[0104] The term “antigen-binding fragment,” as used herein, refers to one or more fragments of an immunoglobulin that retain the ability to specifically bind to a target antigen. The antigen-binding function of an immunoglobulin can be performed by fragments of a full-length antibody. The antibody fragments can be a Fab, F(ab’)2, scFv, SMIP, diabody, a triabody, an affibody, a nanobody, an aptamer, or a domain antibody. Examples of binding fragments encompassed by the term “antigen-binding fragment” of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb (Ward et al., Nature 341 :544-546, 1989) including VH and VL domains; (vi) a dAb fragment that consists of a VH domain; (vii) a dAb that consists of a VH or a VL domain; (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv)). These antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments can be screened for utility in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in certain cases, by chemical peptide synthesis procedures known in the art. PATENT

[0105] ATTORNEY DOCKET NO.: 51772-016WO2

[0106] As used herein, “percent (%) sequence identity” with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity values may be generated using the sequence comparison computer program BLAST. As an illustration, the percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which can alternatively be phrased as a given nucleic acid or amino acid sequence, A that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows:

[0107] 100 multiplied by (the fraction X / Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program’s alignment of A and B, and where Y is the total number of nucleic acids in B. It will be appreciated that where the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0108] As used herein, the term “vector” includes a nucleic acid vector, e.g., a DNA vector, such as a plasmid, an RNA vector, virus, or other suitable replicon (e.g., viral vector). A variety of vectors have been developed for the delivery of polynucleotides encoding exogenous proteins into a prokaryotic or eukaryotic cell. Examples of such expression vectors are disclosed in, e.g., WO 1994 / 011026; incorporated herein by reference as it pertains to vectors suitable for the expression of a gene of interest. Expression vectors suitable for use with the compositions and methods described herein contain a polynucleotide sequence as well as, e.g., additional sequence elements used for the expression of proteins and / or the integration of these polynucleotide sequences into the genome of a mammalian cell. Certain vectors that can be used for the expression of transgenes as described herein include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription. Enhancers serve as binding sites for transcription factors and other regulators which help with DNA looping and recruitment of transcriptional machinery to promoters. Other useful vectors for expression of a transgenes contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements include, e.g., 5’ and 3’ untranslated regions, an internal ribosomal entry site (IRES), and polyadenylation signal (pA) site in order to direct efficient transcription of the gene carried on the expression vector. The expression vectors suitable for use with the compositions and methods described herein may also contain a polynucleotide encoding a marker for selection of cells that contain PATENT

[0109] ATTORNEY DOCKET NO.: 51772-016WO2 such a vector. Examples of a suitable marker are genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, nourseothricin, or zeocin.

[0110] As used herein, the terms “adeno-associated virus” and “AAV” include, but are not limited to, AAV type 1 , AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11 , AAV type 12, AAV type 13, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, and any other AAV now known or later discovered. See, e.g., Fields et al. Virology, 4thed. Lippincott-Raven Publishers, Philadelphia, 1996. Additional AAV serotypes and clades have been identified recently. (See, e.g., Gao et al. J. Virol. 78:6381 (2004); Moris et al. Virol. 33:375 (2004). The genomic sequences of various serotypes of AAV, as well as the sequences of the native inverted terminal repeats (ITRs), Rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Numbers NC — 002077, NC — 001401 , NC — 001729, NC— 001863, NC— 001829, NC— 001862, NC— 000883, NC— 001701 , NC— 001510, NC— 006152, NC— 006261 , AF063497, U89790, AF043303, AF028705, AF028704, J02275, J01901 , J02275, X01457, AF288061 , AH009962, AY028226, AY028223, AY631966, AX753250, EU285562, NC— 001358, NC — 001540, AF513851 , AF513852 and AY530579; the disclosures of which are incorporated by reference herein for teaching AAV nucleic acid and amino acid sequences. See also, e.g., Bantel-Schaal et al. J. Virol. 73:939 (1999); Chiorini et al. J. Virol. 71 :6823 (1997); Chiorini et al. J. Virol. 73:1309 (1999); Gao et al. Proc. Nat. Acad. Sci. USA 99:11854 (2002); Moris et al. Virol. 33:375 (2004); Muramatsu et al. Virol. 221 :208 (1996); Ruffing et al. J. Gen. Virol. 75:3385 (1994); Rutledge et al. J. Virol. 72:309 (1998); Schmidt et al. J. Virol. 82:8911 (2008); Shade et al. J. Virol. 58:921 (1986); Srivastava et al. J. Virol. 45:555 (1983); Xiao et al. J. Virol. 73:3994 (1999); WO 00 / 28061 , WO 99 / 61601 , WO 98 / 11244; and US 6,156,303; the disclosures of which are incorporated by reference herein for teaching AAV nucleic acid and amino acid sequences.

[0111] From a structural perspective, AAV is a nonpathogenic parvovirus composed of a 4.7 kb singlestranded DNA genome within a non-enveloped, icosahedral capsid. The genome contains three open reading frames (ORF) flanked by inverted terminal repeats (ITR) that function as the viral origin of replication and packaging signal. The Rep ORF encodes four nonstructural proteins that play roles in viral replication, transcriptional regulation, site-specific integration, and virion assembly. The Cap ORF encodes three structural proteins (VP 1 -3) that assemble to form a 60-mer viral capsid. Finally, an ORF present as an alternate reading frame within the Cap gene produces the assembly-activating protein (AAP), a viral protein that localizes AAV capsid proteins to the nucleolus and functions in the capsid assembly process. There are several naturally occurring ("wild-type") serotypes and over 100 known variants of AAV, each of which differs in amino acid sequence, particularly within the hypervariable regions of the capsid proteins, and thus in their gene delivery properties. No AAV has been associated with any human disease, making recombinant AAV attractive for clinical applications.

[0112] The genomic sequences of various serotypes of AAV, as weii as the sequences of the native terminal repeats (TRs), Rep proteins, and capsid subunits, are known in the art. Such sequences may be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Numbers NC__002077.1 (AAV1 ), AF063497.1 (AAV1 ), NC_001401.2 (AAV2), AF043303.1 (AAV2), J01901 .1 PATENT

[0113] ATTORNEY DOCKET NO.: 51772-016WO2

[0114] (AAV2), U48704.1 (AAV3A), NC 001729.1 (AAV3A), AF028705.1 (AAV3B), NC .001829.1 (AAV4), U89790.1 (AAV4), NC..006152.1 (AA5), AF085716.1 (AAV-5), AF028704.1 (AAV6), NC 006260.1 (AAV7), AF513851 .1 (AAV7), AF513852.1 (AAV8) NC 006261 .1 (AAV-8), AY530579.1 (AAV9), AAT46337 (AAV10) and AAO88208 (AAVrhI O); the disclosures of which are incorporated by reference herein for teaching AAV nucleic acid and amino acid sequences. See also, e.g., Srivistava et ai. (1983) J. Virology 45:555: Ch iorini et al. (1998) J. Virology 71 :6823; Chiorini et al. (1999) J. Virology 73: 1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996) Virology 221 :208; Shade et al. (1986) J. Virol. 58:921 ; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99: 11854; Moris et al. (2004) Virology 33:375-383; international patent publications WO 00 / 28061 , WO 99 / 61601 , WO 98 / 11244; and U.S. Pat. No. 6,156,303.

[0115] As used herein, the term "packaging" refers to a series of intracellular events that result in the assembly and encapsidation of an AAV particle. AAV “Rep” and “Cap” genes refer to polynucleotide sequences encoding replication and encapsidation proteins of adeno-associated virus. AAV Rep and Cap are referred to herein as AAV “packaging genes”.

[0116] “Rep proteins” are proteins which fill capsids with a polynucleotide molecule (e.g., viral DNA) into a capsid. In addition, a Rep protein may enable nucleic acid molecule replication, transcriptional regulation of a nucleic acid molecule, and / or site-specific integration (e.g., chromosomal integration) of a nucleic acid molecule. The Rep protein may be from any parvovirus. As one of skill in the art will appreciate, in some parvovirus family virus species, a Rep protein is referred to as a “non-structural (NS) protein”. As used herein, a Rep protein may refer to an NS protein. As used herein, a Rep protein may refer to an AAV Rep protein, which has been found in all AAV serotypes examined to date, and a synthetic Rep protein. For example, in some embodiments, an AAV Rep protein (e.g., AAV Rep40, AAV Rep52, AAV Rep68, and AAV Rep78) is a capsid protein having an amino acid sequence derived from a particular AAV serotype, for example AAV type 1 , AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11 , AAV type 12, AAV type 13, avian AAV, bovine AAV, canine AAV, goat AAV, snake AAV, equine AAV, and ovine AAV. Alternatively, for example, as used herein, a Rep protein may include a synthetic Rep protein. Any suitable Rep protein may be used.

[0117] As used herein, the term “viral capsid protein” refers to a capsid protein composing a proteinaceous shell. Such a proteinaceous shell is generally composed of one or more viral capsid proteins and when assembled is capable of being loaded with one or more polynucleotide molecules. A viral capsid protein described herein may, for example, be a viral protein VP1 , VP2, or VP3. Further, a viral capsid protein described herein may refer to a synthetic protein or a viral capsid protein from Parvoviridae (e.g., an AAV). A “viral capsid protein” as used herein refers to any of the AAV capsid proteins that are components of AAV viral particles.

[0118] As used herein, the terms “viral protein 1 ” and “VP1” refer to any capsid protein that is a component of a capsid, for example, a parvovirus (e.g., AAV) capsid particle. As used herein, a VP1 may possess a surface binding site that interacts with one or more molecules on the surface of a cell to initiate the process of cell entry (e.g., endocytic entry and receptor-mediated fusion). As used herein, a VP1 may self-assemble into a structure consisting of VP1 , VP2, and / or VP3 molecules. VP1 may exhibit self- PATENT

[0119] ATTORNEY DOCKET NO.: 51772-016WO2 binding properties and self-assemble around the exterior of a respective VP1 -containing capsid. As used herein, a VP1 may be synthetic or a VP1 derived from Parvoviridae (e.g., an AAV). For example, a VP1 derived from an AAV may be a VP1 derived from AAV type 1 , AAV type 2, AAV type 3, AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11 , AAV type 12, AAV type 13, avian AAV, bovine AAV, canine AAV, goat AAV, snake AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, or any other AAV now known and later discovered.

[0120] As used herein, the terms “encapsidation,” “encapsulating,” “encapsidate,” and the like refer to non-enzymatically driven encasing of nucleic acid molecules in a capsid shell. Thus, when a suitable population of viral capsid proteins (e.g., VP1 , VP2, and / or VP3) and conditions are combined with a nucleic acid molecule, the nucleic acid molecule may be encapsidated by the capsid to form an assembled capsid particle packaged with one or more nucleic acid molecules. The encapsidation process may occur simultaneously with capsid assembly or after capsid assembly is complete.

[0121] An “ITR” is a palindromic nucleic acid, e.g., an inverted terminal repeat, that is about 120 nucleotides to about 250 nucleotides in length and capable of forming a hairpin. The term “ITR” includes the site of the viral genome replication that may be recognized and bound by a parvoviral protein (e.g., Rep78 / 68). An ITR may be from any AAV, with serotype 2 being preferred. An ITR includes a replication protein binding element (RBE) and a terminal resolution sequence (TRS). The term “ITR” does not require a wild-type parvoviral ITR (e.g., a wild-type nucleic acid sequence may be altered by insertion, deletion, truncation, or missense mutations), as long as the ITR functions to mediate virus packaging, replication, integration, and / or provirus rescue, and the like. The “5’ ITR” is intended to mean the parvoviral ITR located at the 5’ boundary of the nucleic acid molecule; and the term “3’ ITR” is intended to mean the parvoviral ITR located at the 3’ boundary of the nucleic acid molecule.

[0122] As used herein, the term "helper virus" for AAV refers to a virus that allows AAV (e.g., wild-type AAV) to be replicated and packaged by a mammalian cell. A variety of such helper viruses for AAV are known in the art, including adenoviruses, herpesviruses, and poxviruses such as vaccinia. The adenoviruses encompass several different subgroups, although Adenovirus type 5 of subgroup C is most commonly used. Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC. Viruses of the herpes family include, for example, herpes simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are also available from depositories such as ATCC.

[0123] As used herein, the term "helper virus functions" refers to functions encoded in a helper virus genome which allow AAV replication and packaging (in conjunction with other requirements for replication and packaging described herein). As described herein, "helper virus function" may be provided in several ways, including by providing helper virus or providing, for example, polynucleotide sequences encoding the requisite function(s) to a producer cell in trans. For example, a plasmid or other expression vector comprising nucleotide sequences encoding one or more adenoviral proteins is transfected into a producer cell along with an rAAV vector. The terminology "infectious" virus or viral particle is one that comprises a competently assembled viral capsid and is capable of delivering a polynucleotide component into a cell for which the viral species is tropic. The term does not necessarily imply any replication capacity of the virus. Assays for counting infectious viral particles are described elsewhere in this disclosure and in the PATENT

[0124] ATTORNEY DOCKET NO.: 51772-016WO2 art. Viral infectivity may be expressed as the ratio of infectious viral particles to total viral particles. Methods of determining the ratio of infectious viral particle to total viral particle are known in the art. See, e.g., Grainger et al. (2005) Mol. Ther. 11 : S337 (describing a TCID50 infectious titer assay); and Zolotukhin et al. (1999) Gene Ther. 6:973.

[0125] As used herein, the term “transgene” refers to a recombinant nucleic acid (e.g., DNA or cDNA) encoding a gene product (e.g., a therapeutic gene product). The gene product may be an RNA, a peptide, a polypeptide, or a protein. In addition to the coding region for the gene product, the transgene may include or be operably linked to one or more elements to facilitate or enhance expression, such as a promoter, enhancer(s), destabilizing domain(s), response element(s), reporter element(s), insulator element(s), polyadenylation signal(s) and / or other functional elements. Embodiments may utilize any known suitable promoter, enhancer(s), destabilizing domain(s), response element(s), reporter element(s), insulator element(s), polyadenylation signal(s), and / or other functional elements.

[0126] As used herein, the terms “wild-type,” “naturally occurring,” or “non-mutant” form of a gene refers to a nucleic acid that encodes a protein associated with normal or non-pathogenic activity (e.g., a protein lacking a mutation). In some embodiments, the wild-type gene may serve as a reference to compare a variant gene that is associated with a genetic disorder, e.g., as described in Table 3 herein.

[0127] As used herein, the term “variant” or “mutant” refers to any gene with a change in sequence, such that the sequence is not identical to that of the wild-type gene and results in an altered form of the gene. A mutation may be selected from the group including a single nucleotide point mutation that results in a premature termination codon, a single nucleotide insertion, a single nucleotide deletion, the insertion of two or more contiguous nucleotides, the deletion of two or more contiguous nucleotides, the duplication of a contiguous region within a gene (e.g., an exon), or the deletion of a contiguous region within a gene. A mutated gene may include a single mutation, or multiple mutations. A mutation may occur in any region of the gene. Gene mutations include the substitution, insertion, or deletion of a single base in DNA or the substitution, insertion, deletion, or rearrangement of multiple bases or larger sections of genes or chromosomes, including repeat expansions.

[0128] As used herein, the terms “hybridization,” “annealing,” or “binding” of nucleic acids is achieved when one or more nucleoside residues within a polynucleotide base pairs with one or more complementary nucleosides to form a stable duplex. The base pairing is typically driven by hydrogen bonding events. Hybridization includes Watson-Crick base pairs formed from natural and / or modified nucleobases. The hybridization can also include non-Watson-Crick base pairs, such as wobble base pairs (guanosine-uracil, hypoxanthine-uracil, hypoxanthine-adenine, and hypoxanthine-cytosine) and Hoogsteen base pairs. Nucleic acids need not be 100% complementary to undergo hybridization. For example, one nucleic acid may be, e.g., 95% complementary, 90%, complementary, 85% complementary, 80% complementary, 75% complementary, 70% complementary, 65% complementary, 60% complementary, 55% complementary, 50% complementary, or less, relative to another nucleic acid, but the two nucleic acids may still form sufficient base pairs with one another so as to hybridize.

[0129] As used herein, the phrase “operably linked” refers to a functional connection between two or more molecules, constructs, transcripts, entities, moieties, or the like. For example, one segment of DNA may be operably linked to another segment of DNA if they are positioned relative to one another on the PATENT

[0130] ATTORNEY DOCKET NO.: 51772-016WO2 same contiguous DNA molecule and have a structural or functional relationship, such as a promoter or enhancer that is positioned relative to a coding region to facilitate transcription of the coding region. In other examples, the operably linked nucleic acids are not contiguous, but are positioned in such a way that they have a functional relationship with each other as nucleic acids or as proteins that are expressed by them. Enhancers, for example, do not have to be contiguous. Linking may be accomplished by ligation at convenient restriction sites or by using synthetic oligonucleotide adaptors or linkers.

[0131] As used herein, the term “contacting” (i.e., contacting a cell with an agent) is intended to include incubating the agent and the cell together in vitro (e.g., adding the agent to cells in culture) or administering the agent to a subject such that the agent and cells of the subject are contacted in vivo. The term “contacting” is not intended to include exposure of cells to an agent that may occur naturally in a subject (i.e., exposure that may occur as a result of a natural physiological process).

[0132] As used herein, the terms “associated with,” conjugated,” “linked,” “attached,” and “tethered,” when used with respect to two or more moieties, means that the moieties are physically associated or connected with one another, either directly or via one or more additional moieties that serves as a linking agent, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions. An “association” need not be strictly through direct covalent chemical bonding. It may also suggest ionic or hydrogen bonding or a hybridization-based connectivity sufficiently stable such that the “associated” entities remain physically associated.

[0133] As used herein “modified” refers to a changed state or structure of a molecule (e.g., a polynucleotide (e.g., DNA or mRNA); e.g., a polypeptide or protein (e.g., an amino acid residue)) of the invention. Molecules may be modified in many ways, such as structural modifications (e.g., mutation of one or more base pairs or amino acid residues) or chemical modifications (e.g., methylation, acetylation, reduction or oxidation, glycosylation, lipidation, ubiquitination, of one or more base pairs or amino acid residues). In some embodiments, a molecule such as a DNA or an mRNA is modified to remove, reduce, or eliminate DRACH motifs to reduce the number of m6A methylation modifications in a gene or coding sequence of interest.

[0134] As used herein, the term “express” or “expression” refers to one or more of the following events: (1 ) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and / or 3' end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein. Expression of a gene of interest in a subject can manifest, for example, by detecting: an increase in the quantity or concentration of mRNA encoding a corresponding protein (as assessed, e.g., using RNA detection procedures such as quantitative polymerase chain reaction (qPCR) and RNA sequencing (RNA-seq) techniques, among other RNA detection methods known in the art), an increase in the quantity or concentration of a corresponding protein (as assessed, e.g., using protein detection methods described herein or known in the art, such as enzyme-linked immunosorbent assays (ELISA), Western blot, or mass spectrometry, among others), and / or an increase in the activity of a corresponding protein (e.g., in the case of an enzyme, as assessed using an enzymatic activity assay known in the art) in a sample obtained from the subject. PATENT

[0135] ATTORNEY DOCKET NO.: 51772-016WO2

[0136] As used herein, the term “gene therapy agent” refers to a composition that contains a nucleic acid encoding a therapeutic gene of interest and that is formulated in such a way as to facilitate expression of the gene upon administration to a desired subject. Exemplary gene therapy agents include viral vectors harboring genomes containing nucleic acids that encode the therapeutic gene of interest (e.g., adeno- associated virus (AAV) vectors, lentiviral vectors, adenoviral vectors, vaccinia viral vectors, and baculoviral vectors, among others). Additional examples of gene therapy agents include recombinant cells that have been engineered to express the gene of interest. Examples of such cell therapy modalities include stem cells (e.g., pluripotent stem cells, adult stem cells, embryonic stem cells, cancer stem cells, and CD34+ stem cells), among others described herein and known in the art. Such gene therapy agents are administered to a patient with the aim of inducing the expression of the desired gene, thereby providing a therapeutic effect.

[0137] As used herein, the terms “eliminate,, “elimination,” “eliminating,” “terminate,” “termination,” “terminating,” “silence,” “silencing,” and the like, in the context of gene expression, refer to a reduction of gene expression such that the amount or concentration of the product encoded by the corresponding gene falls to a level that is less than that which is necessary to exert its intended therapeutic effect in a subject (i.e., a level that is beneath the therapeutic window of the product encoded by the corresponding gene). These terms can be used interchangeably to refer to termination of gene expression. Termination of gene expression (e.g., termination of the expression of a transgene encoding a protein of interest, such as a transgene previously administered as part of a recombinant AAV vector, as described herein) may be induced by an exogenous agent, such as a Cre recombinase as described herein. Termination of gene expression may be confirmed or quantified by any suitable method for measuring the expression of a nucleic acid or a protein (including protein activity) known in the art or described herein, in which a sample in which gene expression is terminated is compared to an equivalent reference sample (e.g., a sample obtained prior to administration of Cre recombinase). An observation that the level of expression of the encoded gene product has fallen to a level that is beneath the therapeutic window of the gene product may be taken as an indication that expression of the corresponding gene has been “eliminated,” “terminated,” “silenced,” or “turned off,” as those terms are used herein.

[0138] As used herein, the term “gene expression silencer” refers to an agent, composition, molecule, nucleic acid, polypeptide, or protein that reduces gene expression such that the expression level of the gene falls to a level that is beneath that which is necessary to elicit the originally intended therapeutic phenotype (i.e., such that the expression level of the gene falls to a level that is beneath the therapeutic window of the product encoded by the gene). In some embodiments, the gene expression silencer is a recombinase, such as a Cre recombinase described herein, which may catalyze site-specific recombination between recombinase-recognition sequences that flank the gene of interest (and / or a gene expression regulatory element associated therewith). Upon catalyzing this recombination event, the recombinase is deemed to have reduced, eliminated, or fine-tuned expression of the corresponding gene.

[0139] As used herein in the context of a recombinase that is administered to a subject, the term “subsaturating dose” refers to a quantity or concentration of the recombinase that is less than that required to terminate expression of a target transgene (i.e., a quantity or concentration that is less than that required to reduce expression of the target transgene to beneath the therapeutic window of the product encoded PATENT

[0140] ATTORNEY DOCKET NO.: 51772-016WO2 by the target transgene), but that is sufficient, either in isolation or in combination with one or more additional, subsequent doses, to down-titrate the expression level of a target transgene to within its therapeutic window. In contrast, a “saturating dose” of a recombinase refers to a quantity or concentration of the recombinase that is sufficient to terminate expression of a target transgene (i.e., a quantity or concentration that is sufficient to reduce expression of the target transgene to beneath the therapeutic window of the product encoded by the target transgene).

[0141] As used herein, the term “stability” in reference to biological material or a molecule (e.g., a polynucleotide or a polypeptide) refers to the balance of production (e.g., transcription or translation) and decay or degradation or the steady-state levels of the biological material in a system, such as a whole organism, an organ, a tissue or subset of tissues, a cell or a subset of cells, or in a dish or receptacle. In some embodiments, stability refers to the half-life of the biological material or molecule.

[0142] As used herein, the term "exogenous" describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is not found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell). Exogenous materials include those that are provided from an external source to an organism or to cultured matter extracted therefrom.

[0143] As used herein, the term “in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).

[0144] As used herein, the term “in vivo" refers to events that occur within an organism (e.g., animal, plant, microbe, cell, or tissue thereof).

[0145] As used herein, the term “ex vivo" refers to events that occur to a component of an organism (e.g., a tissue, a cell, or a subcellular fraction) when it is taken from the natural environment (e.g., from the body or natural structure) and placed into an artificial environment (e.g., a test tube or a culture dish, flask, or other receptacle) for experimental or clinical applications. In some instances, ex vivo experimentation or applications may involve administering (e.g., implanting, injecting, depositing, infusing, among other suitable routes of administration) the component to the same subject or a separate recipient subject following one or more ex vivo applications.

[0146] As used herein, the terms “treat”, “treatment”, or “treating” in reference to a disease or condition, refer to an approach for obtaining beneficial or desired results, e.g., clinical results. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions; diminishment of extent of disease or condition; stabilized (i.e., not worsening) state of disease, disorder, or condition; preventing spread of disease or condition; delay or slowing the progress of the disease or condition; amelioration or palliation of the disease or condition; and remission (whether partial or total), whether detectable or undetectable. “Ameliorating” or “palliating” a disease or condition means that the extent and / or undesirable clinical manifestations or symptoms of the disease, disorder, or condition are lessened and / or time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those PATENT

[0147] ATTORNEY DOCKET NO.: 51772-016WO2 already with the condition or disorder, as well as those prone to have or at risk of having the condition or disorder (e.g., for prophylactic treatment).

[0148] As used herein, the term “therapeutic protein” refers to (i) a protein whose deficiency or lack of activity is associated with a disorder (e.g., a genetic disorder, for example, a loss-of-function disorder recited in Table 3), as well as (ii) a protein that is not necessarily deficient in a patient, but whose expression or supplementation would nonetheless have a beneficial effect on the patient.

[0149] As used herein, the term “therapeutic window” refers to a range of in vivo concentrations of a therapeutic agent (e.g., a therapeutic protein) in a subject, within such range the therapeutic agent is both: (i) efficacious in treating the disease or condition of interest for which the therapeutic agent is indicated, and (ii) safely tolerated by the subject. Accordingly, the “therapeutic window” for a given therapeutic agent is a range of in vivo concentrations of the therapeutic agent within a subject that begins with (a) the minimum concentration of the therapeutic agent that is effective in treating the corresponding disease or condition (e.g., as assessed by monitoring for the reduction in frequency and / or severity of one or more symptoms thereof), and ends with (b) the maximum concentration that is tolerated by the subject, beyond which one or more adverse events are observed. Exemplary adverse events that may be taken as an indication that the upper bound of a therapeutic window has been surpassed include, without limitation, an undesired immunogenic response mounted by the subject following administration of the therapeutic agent (e.g., an immunogenic response that is specifically directed against the therapeutic agent, as assessed, for instance, by detecting a B cell population, T cell population, or antibody population that specifically binds the therapeutic agent), as well as a manifestation of any off-target effect that is associated with, or attributable to, the therapeutic agent.

[0150] As used herein, the term “sample” refers to a subset of its tissues, cells or component parts (e.g. body fluids, including but not limited to peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood). A sample further may include a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs. A sample further refers to a medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins or nucleic acid molecules.

[0151] As used herein, the term “biodistribution” in reference to a nucleic acid refers to the location of the nucleic acid within a subject upon administration of the nucleic acid to the subject. Biodistribution may occur naturally, e.g., based on physiological factors within the subject. A nucleic acid may be modified, e.g. a recombinant nucleic acid, such that the nucleic acid “biodistributes” to specific cell types, tissues, tissue systems, organs, or organ systems. PATENT

[0152] ATTORNEY DOCKET NO.: 51772-016WO2

[0153] As used herein, the term “reference level” herein refers to a value from a “reference sample” or a “control sample” to determine the effect induced by the methods described herein. A reference level may be a metric or measurement determined prior to administration or implementation of the method (e.g., one or more codon optimization methods or one or more methods of treatment described herein). A reference level may be a metric or measurement determined in a reference sample in which the methods described herein were not administered (e.g., a negative control sample (e.g., a healthy subject control or a subject with a disease or condition)). A reference level may be a metric or measurement determined in a reference sample that exhibits a known or expected effect in order to evaluate the efficacy of the methods described herein (e.g., effects produced by routine methods of protein production, or effects produced by known methods of treatment). In some embodiments, a reference level may be a predetermined value or a value. As the skilled artisan will appreciate, the reference level is predetermined and set to meet the requirements in terms of, for example, specificity and / or sensitivity. It may be, for example, that assay sensitivity or specificity, respectively, has to be set to certain limits, e.g., 80%, 90%, or 95%. These requirements may also be defined in terms of positive or negative predictive values. In one embodiment, the reference level is determined in healthy individuals. The reference value in one embodiment has been predetermined in the disease entity to which a subject belongs. In certain embodiments, the reference level can be set to any percentage between, e.g., 25% and 75% of the overall distribution of the values in a disease entity investigated. In other embodiments, the reference level can be set to, for example, the median, tertiles, quartiles, or quintiles as determined from the overall distribution of the values in a disease entity investigated or in a given population. In one embodiment, the reference level is set to the median value as determined from the overall distribution of the values in a disease entity investigated. In some embodiments, the reference level may depend on the sex of the patient, e.g., males may have a different reference level than females.

[0154] As used herein, the terms “effective amount,” “therapeutically effective amount,” and a “sufficient amount” of a composition described herein refer to a quantity sufficient to, when administered to the subject (e.g., a mammal such as a human subject) yield beneficial or desired results, including clinical results, and, as such, an “effective amount” or synonym thereto depends upon the context in which it is being applied. The amount of a given composition described herein that will correspond to such an amount will vary depending upon an assortment of factors, such as the given therapeutic agent (e.g., a polynucleotide or a vector; e.g., an AAV comprising a transgene as described herein or a plurality thereof), the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject (e.g., age, sex, weight) or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art. Also, as used herein, a “therapeutically effective amount” of a composition of the present disclosure is an amount which results in a beneficial or desired result in a subject as compared to a reference (e.g., the subject prior to treatment, a healthy control, or an untreated subject). As defined herein, a therapeutically effective amount of a composition of the present disclosure may be readily determined by one of ordinary skill by routine methods known in the art.

[0155] As used herein, the term “therapeutic level,” in reference to protein expression, refers to a protein expression level that is within an acceptable range to confer a desired effect from a treatment (e.g., a level to treat a disease or disorder, as the term “treat” is used herein). A therapeutic level may refer to a PATENT

[0156] ATTORNEY DOCKET NO.: 51772-016WO2 physiologically acceptable expression level based on a healthy control subject, or a median expression level based on a plurality of healthy control subjects. A therapeutic level may refer to an expression level that falls within a range of accepted expression levels based on subject characteristics such as, e.g., a subject’s age, sex, weight, height, ethnicity, lifestyle, and / or one or more comorbidities. A therapeutic level may refer to an expression level that exceeds physiological values, such that the therapeutic expression level has about a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, 500%, or more than about 500% greater relative to an expression level based on a healthy control subject or a median expression level based on a plurality of healthy control subjects.

[0157] As used herein, the term “sub-therapeutic level,” in reference to protein expression, refers to a protein expression level that is below a therapeutic level. A sub-therapeutic level of protein expression may refer to an expression level below a physiologically acceptable or effective expression level based on a healthy control subject or a median expression level based on a plurality of healthy control subjects. A sub-therapeutic level may refer to an expression level that falls within a range of accepted expression levels based on subject characteristics such as, e.g., a subject’s age, sex, weight, height, ethnicity, lifestyle, and / or one or more comorbidities. A sub-therapeutic level may refer to an expression level that is below a physiological value, such that the sub-therapeutic expression level is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% lower than a physiological value. A sub-therapeutic level may refer to an expression level that is below a therapeutic level, such that the sub-therapeutic expression level is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% lower than a therapeutic level.

[0158] As used herein, the term ’’safely tolerated level,” in reference to protein expression, refers to a protein expression level (e.g., expression of a protein of interest encoded by a transgene) that does not induce an adverse reaction that would be considered harmful or unacceptable by a physician of skill in the art. Exemplary adverse events that may include, without limitation, an undesired immunogenic response mounted by a subject to the encoded protein product (e.g., an immunogenic response that is specifically directed against the encoded protein product, as assessed, for instance, by detecting a B cell population, T cell population, or antibody population that specifically binds the therapeutic agent), as well as a manifestation of any off-target effect (e.g., toxicity) that is associated with, or attributable to, the therapeutic agent. A safely tolerated level for a protein of interest in a subject may depend on the subject’s condition, as described herein. As used herein, “administration” refers to dispensing, delivering, or applying a composition of the disclosure to a subject by any suitable route for delivery of the composition (e.g., a polynucleotide or a vector such as an AAV comprising a transgene as described herein or a plurality thereof), to the desired location in the subject. Exemplary routes of administration include intramuscular, subcutaneous, intradermal, intravenous, intraperitoneal, intraarterial, intraventricular, intranasal, intraorbital, intracranial, or intraosseous administration.

[0159] As used herein, the term “administered in combination” or “combined administration” means that two or more agents are administered to a subject at the same time or within an interval such that there may be an overlap of an effect of each agent on the subject in need thereof. In some embodiments, the PATENT

[0160] ATTORNEY DOCKET NO.: 51772-016WO2 agents are administered within about 1 or more weeks, 1 or more days, 1 or more hours, or 1 or more minutes of one another. In some embodiments, the administrations of the agents are spaced sufficiently closely together such that a combinatorial (e.g., a synergistic) effect is achieved.

[0161] As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues a subject, such as a mammal (e.g., a human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0162] As used herein, the term “pharmaceutically acceptable excipient,” as used herein, refers to any ingredient other than active agents (e.g., as described herein) present in pharmaceutical compositions and having the properties of being substantially nontoxic and non-inflammatory in subjects.

[0163] As used herein, the term “pharmaceutical composition” refers to a mixture containing a therapeutic agent, optionally in combination with one or more pharmaceutically acceptable excipients, diluents, and / or carriers, to be administered to a subject, such as a mammal (e.g., a human) in order to treat, reduce the likelihood of, or otherwise control a particular disease or condition affecting or that may affect the subject.

[0164] As used herein, the term "heterologous" means derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked is a heterologous promoter. Thus, for example, an rAAV that includes a heterologous nucleic acid sequence encoding a heterologous gene product is an rAAV that includes a polynucleotide not normally included in a naturally occurring wild-type AAV, and the encoded heterologous gene product is a gene product not normally encoded by a naturally occurring wildtype AAV.

[0165] A “promoter” is a nucleic acid enabling the initiation of the transcription of a gene in a messenger RNA, such transcription being initiated with the binding of an RNA polymerase on or nearby the promoter.

[0166] As used herein, the term “lipid nanoparticle” or “LNP” refers to a transfer or delivery vehicle including one or more lipids (e.g., cationic lipids, non-cationic lipids, and PEG-modified lipids). LNPs are nanoparticles composed of lipids. LNPs are generally spherical and have an average diameter ranging from 10 to 1000 nm. Examples of suitable lipids include, for example, the phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides). Examples of suitable lipids also include, for example, triglycerides, diglycerides, monoglycerides, fatty acids, steroids, and waxes. LNPs may contain a cationic lipid, or a lipid species with a net positive charge at a selected pH (e.g., physiological pH), to encapsulate and / or enhance the delivery of a therapeutic product into the target cells. In some embodiments, a Ore recombinase of the present disclosure is formulated in an LNP (LNP-Cre). Exemplary LNPs are formulated to deliver Ore recombinase to one or more target cells. LNPs may comprise an ionizable lipid (e.g., an ionizable amino lipid), a sterol or other structural lipid, a non-cationic helper lipid or phospholipid, and a PEG-modified lipid. Emulsifiers or surfactants PATENT

[0167] ATTORNEY DOCKET NO.: 51772-016WO2 stabilize lipid cores in LNPs. Emulsifiers are used to stabilize lipid dispersion and they prevent particle agglomeration. Lipid components of LNPs can help improve LNP properties, such as particle stability, delivery efficacy, tolerability, and biodistribution. Organ selectivity can vary based on the relative proportions of the lipid components and routes of administration.

[0168] As used herein, the term “providing” refers to direct administration of a therapeutic product / agent (e.g., a therapeutic transgene encoding a therapeutic protein) or administration of a precursor that becomes the therapeutic agent in vivo, for example, after being processed in vivo. The therapeutic agent can be, for example, an AAV comprising a transgene which encodes a protein functioning as a precursor (e.g., a transgene for encoding a protein which will be expressed and processed in vivo to become a functional product), an AAV comprising transgene which encodes for a microRNA, an AAV comprising a transgene which encodes for an siRNA, an AAV comprising a transgene which encodes for Cre recombinase, an AAV comprising a polynucleotide comprising a floxed transgene of interest or a floxed transgene expression cassette, or an AAV comprising a transgene which directly encodes the therapeutic protein without needing a precursor.

[0169] As used herein, the term “recombinase” refers to a site-specific enzyme that mediates the recombination of a DNA between recombinase recognition sites, which results in the excision, integration, inversion, or exchange of DNA fragments between the recombinase recognition sequences. In some examples, the recombinase is a Cre recombinase, such as a Cre recombinase having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to the amino acid sequence of a Cre recombinase described herein.

[0170] A recombinase may specifically bind and “cleave” at recombinase recognition sites. As used herein, “cleavage” refers to introduction of a break in a nucleic acid molecule, which may be either a single-stranded break or a double-stranded break. In some embodiments, cleavage by a Cre recombinase introduces a double-stranded break. Cleavage may result in excision, integration, inversion, or exchange of DNA (e.g., a transgene or a functional element within a transgene cassette) between recombinase recognition sites. In some embodiments, the transgene or functional element is excised as a result of cleavage. In other embodiments, the transgene or functional element is ultimately reintegrated into the transgene expression cassette but is rearranged such that its ability to drive expression is reduced or eliminated. In some examples, this rearrangement is inversion of the transgene or functional element, such that the transgene or functional element is oriented in the opposite direction as that required for effective transcription. In some examples, this rearrangement is relocation of the transgene or functional element to a position in the transgene cassette that is no longer conducive to transcription (e.g., placed downstream of the pA signal).

[0171] As used herein, the terms “Cre recombinase”, “Cre”, “Cre enzyme”, and “Cre protein” refer to the Cre recombinase protein encoded by the Cre gene. Cre recombinase is a 38.5 kDa protein with 343 amino acids that form two distinct domains. Cre recombinase is a DNA-binding small bacteriophage P1- derived tyrosine recombinase enzyme. It uses a topoisomerase l-like mechanism to catalyze DNA recombination events between two specific DNA recognition sites, named lox sites, without requiring high-energy cofactors. The terms “Cre recombinase”, “Cre”, “Cre enzyme”, and “Cre protein” as used PATENT

[0172] ATTORNEY DOCKET NO.: 51772-016WO2 herein encompass wild-type Cre protein (SEQ ID NO: 17; UniProt ID: P06956 ■ RECR_BPP1 ) as well as variants of wild-type Cre protein such as variant proteins having at least 85% sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to the amino acid sequence of wild-type Cre recombinase protein (e.g., SEQ ID NO: 17), provided that the variant Cre protein retains the recombinase function of the wild-type Cre protein. As used herein, the terms “Cre recombinase", “Cre”, “Cre enzyme”, and “Cre protein” also encompass proteins encode by the wild-type Cre gene (SEQ ID NO: 20; GenBank: X03453.1 ), as well as proteins encoded by variants of the wild-type Cre gene such as variant polynucleotides having at least 85% sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to the nucleic acid sequence of wild-type Cre gene (e.g., SEQ ID NO: 20).

[0173] As used herein, the term “LNP-Cre” refers to a lipid nanoparticle (LNP)-formulated nucleic acid encoding the Cre recombinase protein or an LNP-formulated Cre recombinase protein.

[0174] As used herein, the term “transgene expression cassette” refers to a polynucleotide that comprises a transgene of interest (e.g., a transgene encoding a therapeutic protein) and other functional components that are essential or useful for gene expression or help regulate gene expression such as a promoter, an enhancer, or a terminator, such as a pA signal. As used herein, the term “functional component” or “functional element” are interchangeable terms for any component that is essential or useful for gene expression or helps regulate gene expression, such as a promoter, an enhancer, or a terminator (e.g., a pA signal).

[0175] As used herein, the term “inactivating” in reference to a transgene refers to reduction of expression of the transgene. “Inactivation” or reduction of expression may be detected either at the level of an individual transgene or as a reduction in the overall expression of the transgene in a population of cells containing the transgene. Within a subject, inactivating a transgene may refer to inactivating a subset of actively expressing transgene copies, such as by a sub-saturating dose, thereby reducing overall therapeutic protein expression levels or activity levels in the subject, e.g. to, to down-titrate the expression level of a target transgene to within its therapeutic window, as described herein.

[0176] As used herein, the terms “recombinase recognition site” and “recombinase recognition sequence” are interchangeable terms in reference to a nucleotide sequence target that is recognized by a recombinase and undergoes strand exchange with another DNA molecule having the recombinase recognition site, resulting in excision, integration, inversion, or exchange of DNA fragments between the recombinase recognition sequences. A recombinase recognition site is recognized by a “corresponding recombinase,” which is said to “correspond” to a specific recognition site if the recombinase either naturally binds to, and catalyzes cleavage at, the recombinase recognition site, or if the recombinase has been artificially modified to bind and catalyze cleavage at the recombinase recognition site. In some examples, the recombinase recognition site is a lox site, such as a lox site having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to the nucleic sequence of a lox site described herein. In some examples, the corresponding recombinase is a Cre recombinase. PATENT

[0177] ATTORNEY DOCKET NO.: 51772-016WO2

[0178] As used herein, the terms “lox” and “lox site” refer to the 34 base pairs (hereinafter “bp”) DNA target sequences that are composed of two 13 bp palindromic regions (recombinase binding elements) flanking an asymmetric 8 bp spacer sequence. The 34-bp target sequence is required for the site-specific recombination process in combination with the Cre protein which mediates the recombination by recognizing the lox sites. In some embodiments, the open reading frame of the gene (e.g., a therapeutic transgene encoding a therapeutic protein) is flanked by the lox sites, thus generating a “floxed” gene. In some embodiments, the “floxed” gene is the one being genetically manipulated by the Cre-lox system. In some embodiments, the transgene expression cassette (or any functional component thereof) is flanked by the lox sites, thus generating a “floxed” transgene expression cassette (or floxed functional component thereof). In some embodiments, the “floxed” transgene expression cassette (or floxed functional component thereof) is the one being genetically manipulated by the Cre-lox system. In some embodiments, the functional component is a promoter, an enhancer, or a terminator (e.g., a pA signal). In some embodiments, the promoter is flanked by the lox sites, thus generating a “floxed” promoter. In some embodiments, the “floxed” promoter is the one being genetically manipulated by the Cre-lox system. In some embodiments, the enhancer is flanked by the lox sites, thus generating a “floxed” enhancer. In some embodiments, the “floxed” enhancer is the one being genetically manipulated by the Cre-lox system. In some embodiments, the terminator is flanked by the lox sites, thus generating a “floxed” terminator. In some embodiments, the “floxed” terminator is the one being genetically manipulated by the Cre-lox system. In some embodiments, the “floxed” terminator is a “floxed” pA signal. In bacteriophage P1 , the lox sites are called loxP sites (locus of crossing over in phage P1). As used herein, the terms “lox” and “lox site” encompass the wild-type 34-bp lox sequences (SEQ ID NO: 21 and its reverse complement SEQ ID NO: 59) as well as variants of the wild-type lox sequence such as variant polynucleotides having at least 85% sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to the nucleic acid sequences of the wild-type lox site (e.g., SEQ ID NO: 21 and its reverse complement SEQ ID NO: 59).

[0179] As used herein, the terms “flank”, “flanking”, and “flanked by” in reference to an element of a vector sequence (e.g., an element within a transgene cassette) means to be located adjacent to said element. “Flanking” elements may be directly adjacent to one another, with no intervening nucleotides, or sufficiently proximal to one another so as for one of the elements to exert its intended effect on the other. For example, recombination recognition sites may “flank” another element even if there are intervening nucleotides, so long as the elements are sufficiently close as to permit the element to undergo recombination (e.g., excision, integration, inversion, or exchange) by virtue of the action of a recombinase on the recombinase recognition sites.

[0180] As used herein, the term “terminator” refers to a nucleic acid sequence within the transgene expression cassette that can reduce transgene expression when expressed. In some embodiments, the terminator is flanked by the lox sites, thus generating a “floxed” terminator. The floxed terminator is in the reverse or inverse orientation compared to the transgene such that the terminator is non-functional and can be inverted by Cre recombinase leading to its expression, thus reducing transgene expression. In some embodiments, the terminator is a pA signal. PATENT

[0181] ATTORNEY DOCKET NO.: 51772-016WO2

[0182] DETAILED DESCRIPTION

[0183] Described herein are compositions and methods for modulating heterologous gene expression. Particularly, the present disclosure provides compositions and methods for recombinase-mediated elimination, reduction, or fine-tuning of the expression of a gene that has previously been provided to a subject by way of gene therapy (e.g., a gene that has previously been provided using an adeno- associated virus (AAV) or another durable gene therapy vector).

[0184] This methodology is beneficial in that it addresses significant challenges that may arise following administration of a gene therapy agent to a subject. The present disclosure is based, at least in part, on the discovery that in certain scenarios, it may be desirable to attenuate expression of a previously provided gene of interest in a subject. This need may arise from any of various underlying causes. The gene therapy agent may have been administered, for instance, in such a manner or quantity that ultimately effectuates transgene expression at a level beyond that which is therapeutically necessary or safely tolerated by the subject. This unintended excess of gene expression may occur systemically, or it may be localized to one or more particular tissues. Additionally or alternatively, the subject may be one for whom the gene therapy agent was indicated when the gene therapy agent was first delivered, but due to subsequent changes in the subject’s condition, the gene therapy agent became contraindicated for the subject at a later date. Specific examples of these scenarios are outlined below, but each case is united by a common theme: the need for a way of reducing, fine-tuning, or eliminating expression of the previously provided transgene. The compositions and methods of the present disclosure meet this challenge, providing the benefit of improving the safety and efficiency of therapeutic genetic therapies.

[0185] In some embodiments, for instance, the gene therapy agent may have been administered to the subject in an amount that results in a level of transgene expression exceeding the therapeutic window of the encoded product. As a particular example, the encoded product of the transgene may be a therapeutic protein, and the gene therapy agent may have been administered in a quantity that results in a total amount or concentration of the encoded protein that inadvertently surpasses the total amount or concentration of the protein that is required to ameliorate the underlying disease or condition being treated. This inadvertently elevated expression of the protein product may occur systemically (e.g., throughout the subject’s body), or the elevated protein expression may be restricted to particular tissues. The compositions and methods of the disclosure may address either of these scenarios, for example, by either reducing expression of the over-expressed gene product in a non-specific manner or by selectively targeting one or more cell types, tissues, organs, or organ systems for attenuating expression of the previously provided gene, thereby positioning the expression level of the gene of interest within the subject’s desired therapeutic window.

[0186] In another example that is similarly applicable to instances in which the encoded product of the transgene is a therapeutic protein, the gene therapy agent may have been administered in a quantity that results in a total amount or concentration of the encoded protein that inadvertently surpasses the total amount or concentration that is safely tolerated by the subject. This arises from the notion that elevated concentrations of a delivered protein may incur the risk of toxicity, for example, due to off-target interactions. As is the case in the foregoing example, this toxicity may be observed systemically (e.g., throughout the subject’s body), or may be restricted to particular tissues. The compositions and methods PATENT

[0187] ATTORNEY DOCKET NO.: 51772-016WO2 of the disclosure can be used to solve either of these two challenges, for example, by either reducing expression of the over-expressed gene product in a non-specific manner or by selectively targeting one or more cell types, tissues, organs, or organ systems for attenuating expression of the previously provided gene. In this way, expression of the gene of interest can be titrated to within a range that is both safe and effective for treating the underlying condition for which the gene of interest was originally intended.

[0188] As a further example, following administration of the gene therapy agent, the subject may develop an immune response to the encoded protein product. This can occur, for instance, if the subject has not had prior exposure to the protein, which may cause cells of the subject’s immune system to mount a humoral or cell-mediated response against the encoded protein, which is being recognized by the subject in this scenario as if the protein were a harmful or foreign antigen. Here again, to mitigate this safety risk, the compositions and methods of the disclosure may reduce expression of the encoded gene therapy product in a non-specific manner or by selectively targeting one or more cell types, tissues, organs, or organ systems for attenuating expression of the previously provided gene. In examples such as this, it may be desirable to reduce expression of the gene of interest to beneath the corresponding therapeutic window, or, alternatively, to a level that is substantially undetectable by way of a given protein expression assay, as the situation dictates.

[0189] In yet another example, following administration of the gene therapy agent, the subject may develop a condition, undergo a lifestyle change, or be exposed to medical interventions (e.g., chronic treatment with a subsequent therapeutic agent) that, either individually or in the aggregate, render the previously administered gene therapy agent contraindicated for the subject. Examples of such conditions that may be developed following administration of the gene therapy agent include, without limitation, a chronic infection (that may necessitate, e.g., administration of an appropriate antiviral, antibiotic, antifungal, or other antimicrobial agent), a cancer or other cell proliferation disorder (that may necessitate, e.g., administration of one or more chemotherapeutic or immunotherapeutic agents), an autoimmune disease (that may necessitate, e.g., administration of an appropriate immunosuppressive agent), or a metabolic condition (that may necessitate, e.g., administration of an agent that promotes or suppresses flux through a particular metabolic cycle), among various other downstream medical conditions that may arise following gene therapy administration. In any of these examples, the newly developed condition and / or the corresponding therapeutic intervention may render the previously provided gene therapy agent contraindicated for the subject. As is the case in the examples recited above, the compositions and methods of the disclosure may address this safety risk by reducing expression of the encoded gene therapy product in a non-specific manner or by selectively targeting one or more cell types, tissues, organs, or organ systems for attenuating expression of the previously provided gene. Here again, it may be desirable to reduce expression of the gene of interest to beneath the corresponding therapeutic window, or, alternatively, to a level that is substantially undetectable by way of a given protein expression assay, as the situation dictates

[0190] From a process perspective, the compositions and methods of the disclosure may be used to address any of the foregoing challenges by leveraging a gene therapy agent that has been designed in a manner that facilitates subsequent excision of the transgene (or a nucleic acid element that regulates transgene expression) from the genetic background of the delivered gene therapy agent. The genetic PATENT

[0191] ATTORNEY DOCKET NO.: 51772-016WO2 background from which this excision occurs may be, for example, the genome of a cell within the subject (as is the case, e.g., for integrative gene therapy agents) or an episomal polynucleotide (as is the case, e.g., for a non-integrative gene therapy agent). In either case, the gene therapy agent may be designed so as to contain recombinase recognition sequences that flank the gene of interest or a component of the gene therapy agent that helps to facilitate expression of the encoded gene product. Following administration of the gene therapy agent to the subject, the excision - and concomitant reduction of gene expression - may be triggered by administering to the subject a recombinase that specifically binds and cleaves the recombinase recognition sites.

[0192] In some embodiments, for example, the gene therapy agent contains a transgene expression cassette that, in turn, encodes a protein of interest, such as a therapeutic protein. In this context, the transgene expression cassette - or a functional component thereof, such as a promoter, enhancer, or the transgene itself, among other elements - may be flanked by a pair of “lox” sites, thereby generating a “floxed” expression cassette (or functional component thereof), in accordance with the definitions of these terms provided above (see “Definitions,” supra). Once the floxed expression cassette or functional component has been delivered to the subject, expression of the protein of interest can then be reduced, as desirable, by administration of a Cre recombinase or a nucleic acid encoding a Cre recombinase. The Cre recombinase may be provided to the subject by way of a variety of delivery techniques, such as through the use of lipid nanoparticles (LNPs), among other modalities described herein. Without being limited by mechanism, once provided to the subject, the Cre recombinase may excise the floxed expression cassette or floxed functional component of the expression cassette, thereby reducing expression of the gene of interest.

[0193] The lox sites described herein may be strategically positioned to flank one or more elements of a transgene expression cassette so as to maximize the reduction in gene expression that is effectuated upon excision of the flanked genetic material.

[0194] For example, in some embodiments, in order to optimally reduce expression of the previously delivered transgene, it may be desirable to excise the entirety of the transgene expression cassette - including the desired transgene (e.g., a therapeutic transgene encoding a therapeutic protein) and the various other functional components of the transgene cassette, such as a corresponding promoter, enhancer, terminator (e.g., a polyadenylation signal), and the like. In scenarios such as this, the entire transgene cassette may be flanked by lox sites, thus generating a floxed transgene expression cassette. Accordingly, delivery of the Cre recombinase would then result in excision of the full transgene expression cassette from its genetic background, thereby reducing expression of the transgene in the subject.

[0195] Alternatively, in some embodiments, lox sites are used to flank one or more non-coding, functional components of the transgene expression cassette. For instance, the functional component(s) may include a promoter, an enhancer, and / or a terminator, such as a polyadenylation (pA) signal. In some embodiments, the promoter is flanked by the lox sites, thus generating a floxed promoter, such that the promoter is the component that is specifically excised by the Cre-lox system. In some embodiments, the enhancer is flanked by the lox sites, thus generating a floxed enhancer, such that the enhancer is the component that is specifically excised by the Cre-lox system. In some embodiments, the terminator is PATENT

[0196] ATTORNEY DOCKET NO.: 51772-016WO2 flanked by the lox sites, thus generating a floxed terminator, such that the terminator is the component that is specifically excised by the Cre-lox system. In some embodiments, the terminator is a pA signal, where the pA signal is flanked by lox sites.

[0197] In some embodiments, the open reading frame of a desired transgene (e.g., a therapeutic transgene encoding a therapeutic protein) can be flanked by lox sites, thus generating a floxed transgene. In this scenario, the transgene is the component that is specifically excised by the Cre-lox system.

[0198] And in still further embodiments, a combination of two or more of the foregoing genetic elements - i.e. , two or more of the transgene of interest, promoter, enhancer, and terminator (e.g., a pA signal), among other functional components of a transgene expression cassette - may be flanked by lox sites, such that subsequent administration of a Cre recombinase results in the excision of this combination of features, thereby reducing expression of the gene of interest.

[0199] Regardless of the component(s) of the transgene expression cassette around which the lox sites are inserted, upon administration of the subsequent Cre recombinase (e.g., by way of delivering the Cre recombinase protein or a nucleic acid encoding the same), the Cre recombinase may catalyze sitespecific recombination between the sequences flanked by the lox sites, thereby reducing, eliminating, or fine-tuning transgene expression. With the appropriate orientation of the lox sites flanking the transgene or transgene expression cassette, transient exposure to the Cre recombinase can result in the excision of the corresponding component(s) of the transgene expression cassette. Any viral or stable non-viral gene expression cassette could be acted upon by a recombinase in this manner to attenuate expression of the transgene product (e.g., a therapeutic protein) previously provided to the subject.

[0200] As is described in the various examples recited above, in some instances, it may be advantageous to reduce, eliminate, or fine-tune transgene expression in a cell type-specific or tissuespecific manner, without affecting or interrupting expression of the transgene systemically. Various methods can be utilized to achieve the targeted attenuation of gene expression that is desired in such cases. For example, in cases in which the Cre enzyme is subsequently delivered to a subject by way of a nucleic acid encoding the same, the open reading frame encoding the Cre enzyme can be placed under the control of a cell type- or tissue-specific gene regulatory element (such as a cell type- or tissue-specific promoter or enhancer, e.g., a muscie-specific promoter and / or enhancer, a liver-specific promoter and / or enhancer, a neuronal-specific promoter and / or enhancer, among others). In this way, attenuation of gene expression can be carried out selectively in the specific cell type or tissue desired (such as in the muscle, liver, and / or neuronal environments), without adversely affecting the expression of the gene of interest in any other cells, tissues, or regions of the subject’s body.

[0201] In some embodiments, the Cre recombinase is administered to the subject in a transient manner (e.g., in one or more doses administered acutely, rather than chronically throughout the subject’s life). In some embodiments, the Cre recombinase is expressed in the subject in a transient manner (e.g., by way of a non-integrative nucleic acid that encodes the Cre recombinase and, after a finite time period, is degraded and cleared from the subject’s body). In some embodiments, the Cre recombinase is administered and / or expressed systemically or in a manner that is restricted to particular cell types or tissue types of interest. PATENT

[0202] ATTORNEY DOCKET NO.: 51772-016WO2

[0203] In specific embodiments, an LNP containing a Cre recombinase or a nucleic acid encoding the same (referred to herein as “LNP-Cre”) can be administered systemically or directly to the muscle (i.e., by way of intramuscular (IM) administration), and may reach the transduced cells in a local manner while limiting system-wide exposure to the recombinase. In other embodiments, the Cre recombinase or nucleic acid encoding the Cre recombinase can be administered by way of a non-LNP delivery technique, such as by way of a viral vector-mediated gene delivery system. Here again, the Cre recombinase may be provided either systemically (e.g., using a viral vector delivery system that is not cell type- or tissuespecific) or directly to muscle cells (e.g., by way of IM administration or a muscle-specific viral vector), such that expression of the recombinase is limited to the muscle environment while limiting system-wide exposure to the recombinase.

[0204] In some embodiments, the floxed transgene or floxed transgene expression cassette is expressed in a tissue other than muscle (e.g., the liver or nervous system, among others) and the Cre recombinase (e.g., LNP-Cre or a viral vector delivery system encoding the Cre recombinase) is administered in a manner that targets such other tissue, thereby limiting system-wide exposure to the recombinase.

[0205] In still other embodiments, the floxed transgene or floxed transgene expression cassette is provided to a subject so as to restrict expression of the transgene to a particular cell type or tissue from the outset of administration of the gene therapy agent. In these instances, for example, the transgene originally administered to the subject may be operably linked to a gene expression regulatory element, such as a promoter or enhancer, that is specifically active in a particular cell type or tissue type. Additionally or alternatively, the transgene originally administered to the subject may be encapsidated by a viral capsid protein (e.g., an AAV capsid protein) that exhibits a tropism for one or more specific cell types or tissue types. In these instances, the subsequently administered Cre recombinase (e.g., LNP-Cre or a viral vector delivery system, among other Cre delivery modalities described herein) may be targeted to a similar set of cell-type or tissue-type distribution as the originally provided transgene. Alternatively, the Cre recombinase (e.g., LNP-Cre or a viral vector delivery system encoding the Cre recombinase) may be provided in a manner that is not restricted to particular cell types or tissue types, particularly when limiting system-wide exposure to the Cre recombinase is not critical due to the already restricted expression profile of the originally provided transgene.

[0206] Importantly, in any of the exemplary gene attenuation scenarios described above or herein, the recombinase enzyme may be provided to the subject either in a single dose or iteratively, such as in a plurality of discrete doses that are separated from one another by way of a desired period of time. The ability to iteratively administer multiple doses of the recombinase enzyme to the subject arises out of a significant benefit that is attributable to certain recombinase delivery vehicles, such as the LNP modality described herein: Unlike certain viral vectors (e.g., AAV vectors), which may elicit an immune response against their corresponding capsid proteins in particular patient populations, LNP-Cre delivery vehicles are substantially non-immunogenic. This discovery has important and advantageous functional consequences.

[0207] First, for example, using the compositions and methods of the disclosure, a subject that has previously been administered a gene therapy agent may be iteratively dosed with a corresponding LNP- PATENT

[0208] ATTORNEY DOCKET NO.: 51772-016WO2

[0209] Cre delivery vehicle with the aim of substantially eliminating expression of the therapeutic transgene (e.g., to a level that no longer effectuates the originally intended phenotype or, alternatively, to an even lower level that is not substantially detectable by way of a desired protein expression assay). By leveraging iterative LNP-Cre dosing, the subject may be administered one dose of a LNP-Cre delivery vehicle described herein, and following a certain period of time (e.g., one or more days, weeks, or months), the subject may be assessed for the expression level of the originally provided transgene of interest. If the expression level has not lowered to the desired threshold (e.g., to a level that is either beneath the therapeutic window of the encoded gene product or to a level that is substantially undetectable, as is desired based on the situation at hand), the subject may be administered one or more subsequent doses of the LNP-Cre delivery vehicle until the desired reduction in transgene expression has been achieved.

[0210] And second, in a similar example, a subject that has previously been administered a gene therapy agent may be iteratively dosed with a corresponding LNP-Cre delivery vehicle with the intention not of eliminating transgene expression, but of down-titrating the expression level of the gene of interest to within the therapeutic window of the encoded gene product. This is particularly useful for those instances described above in which the initial gene therapy agent is provided in an amount that effectuates a level of expression of the transgene that inadvertently surpasses the level of expression that is necessary for achieving the desired phenotype and / or that is safely tolerated without giving rise to toxic side effects. By iteratively dosing the subject with small quantities of an LNP-Cre delivery vehicle, the level of expression of the transgene product may be periodically checked and tuned so as to be brought to within the desired therapeutic and / or safely tolerated range, without the need to eliminate transgene expression altogether.

[0211] The following sections provide a detailed, non-limiting description of various types of transgenes that may be delivered to a subject as part of the subject’s original gene therapy program, as well as nonlimiting examples of various compositions and methods that may be utilized to attenuate expression of the transgene at a later date.

[0212] I. Delivery of a Transgene for Heterologous Expression

[0213] Featured below are various compositions, such as nucleic acid molecules (e.g., transgenes for heterologous expression of a protein of interest or a fragment thereof (e.g., a therapeutic protein or fragment thereof)) and vectors or suitable compositions and methods for delivering said nucleic acid molecules to a host cell. All of these compositions and pharmaceutical compositions thereof and described methods are useful for the methods of treatment described herein.

[0214] A. Transgene for Heterologous Expression

[0215] A transgene for heterologous gene expression may be delivered to a host cell (e.g., a host cell in a subject having or at risk of a disease or condition (e.g., a host cell in a human subject)) for heterologous expression of a polypeptide, a protein, or a fragment thereof (e.g., one or more protein domains, one or more protein chains, or a protein with one or more amino acid deletions). In some embodiments, a transgene for heterologous expression may encode a polypeptide, a protein, or a protein fragment of a PATENT

[0216] ATTORNEY DOCKET NO.: 51772-016WO2 soluble protein, a transmembrane protein, a membrane-associated protein, an intracellular protein, a secreted protein, or a fragment thereof.

[0217] A transgene for heterologous expression may be delivered to a host cell (e.g., a host cell in a subject having or at risk of a disease or condition (e.g., a host cell in a human subject)) as a method of therapy (e.g., gene therapy). In some embodiments, the transgene is identified as a gene that underlies a disease or a condition, such as a gene or a disease or condition set forth in Table 3. In some embodiments, the polypeptide, protein, or fragment thereof is an enzyme (e.g., a protease, a cellulase, a lipase, a hemicellulase, a laccase, an amylase, a glucoamylases, an esterase, a lactase, a polygalacturonase, a galactosidase, a ligninase, an oxidase, a peroxidase, an isomerase, a nitrilase, a hydroxylase, a polymerase, and a depolymerase), a growth factor, an immunomodulator, a cytokine, an antibody, a hormone, a transport protein, a contractile protein, an adhesion protein, a cell junction, or a surface receptor (e.g., an adhesion receptor, a G-protein coupled receptor, a channel, or a transporter). In some embodiments, a transgene for heterologous expression encodes a polypeptide, a protein, or a fragment thereof for ubiquitous or systemic expression (e.g., in all cells, organ systems, or tissues). In some embodiments, a transgene for heterologous expression is expressed in one or more organ systems or a subset of tissues or cell types therein.

[0218] In some embodiments, a transgene encodes a polypeptide, a protein, or a fragment thereof that shares 100% identity to the native protein sequence or a segment of equal length. In some embodiments, a transgene encodes a polypeptide, a protein, or a fragment thereof that shares 75% identity, 76% identity, 77% identity, 78% identity, 79% identity, 80% identity, 81% identity, 82% identity 83% identity, 84% identity, 85% identity, 86% identity, 87% identity, 88% identity, 89% identity, 90% identity, 91% identity, 92% identity, 93% identity, 94% identity, 95% identity, 96% identity, 97% identity, 98% identity, 99% identity to the native protein sequence or a segment of equal length. In some embodiments, a transgene encodes a polypeptide, a protein, or a fragment thereof that is engineered or modified to have one or more mutations associated with enhanced expression, biodistribution, stability, and / or activity (e.g., enhanced binding affinity to one or more binding partners, enhanced catalysis, or enzymatic activity). In some embodiments, a transgene for heterologous expression encodes a fusion protein such as, e.g., an Fc-fusion protein or an albumin-fusion protein, which may further enhance protein stability, protein biodistribution, and / or protein half-life.

[0219] B. Viral Genomes for Delivering a Transgene

[0220] Viral genomes provide a rich source of vectors that can be used for the efficient delivery of a transgene of interest (such as a floxed transgene encoding a therapeutic protein or a floxed transgene expression cassette comprising a transgene encoding a therapeutic protein) into a host cell (e.g., a host cell in a subject having or at risk of a disease or condition (e.g., a host cell in a human subject)). Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the genome of a target cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle and do not require added proteins or reagents to induce gene integration. Examples of viral vectors that may be used in conjunction with the compositions and methods described herein are adeno-associated virus (AAV), retrovirus, PATENT

[0221] ATTORNEY DOCKET NO.: 51772-016WO2 adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses that may be used in conjunction with the compositions and methods described herein include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV- BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). Other examples include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses. Other examples of vectors are described, for example, in US Patent No. 5,801 ,030, the disclosure of which is incorporated herein by reference as it pertains to viral vectors for use in gene therapy.

[0222] AAV Vectors for Delivering a Transgene of Interest

[0223] In some embodiments, a transgene of interest (such as a floxed transgene encoding a therapeutic protein or a floxed transgene expression cassette comprising a transgene encoding a therapeutic protein) is incorporated into recombinant AAV (rAAV) vectors in order to facilitate their introduction into a cell. rAAV vectors useful in the conjunction with the compositions and methods described herein include recombinant nucleic acid constructs that contain (1 ) a polynucleotide such as transgene encoding a protein or fragment thereof and (2) one or more nucleic acids that facilitate expression of the polynucleotide. The viral nucleic acids may include those cis-acting elements of rAAV for replication and packaging (e.g., functional ITRs) of the DNA into a virion. Such rAAV vectors may also contain marker or reporter genes. Useful rAAV vectors include those having one or more of the naturally occurring AAV genes deleted in whole or in part but retain functional flanking ITR sequences. The AAV ITRs may be of any serotype (e.g., derived from serotype 2) suitable for a particular application. Methods for using rAAV vectors are described, for example, in Tai et al. (J. Biomed. Sci. 7:279-291 , 2000), and Monahan and Samulski (Gene Delivery. 7:24-30, 2000), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

[0224] The nucleic acids and vectors described herein can be incorporated into an rAAV virion in order to facilitate introduction of the nucleic acid or vector into a cell. The capsid proteins of AAV compose the exterior, non-nucleic acid portion of the virion and are encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, VP1 , VP2, and VP3, which are required for virion assembly. The construction of rAAV virions has been described, for example, in US Patent Nos. 5,173,414; 5,139,941 ; 5,863,541 ; 5,869,305; 6,057,152; and 6,376,237; as well as in Rabinowitz et al. (J. Virol. 76:791 -801 , PATENT

[0225] ATTORNEY DOCKET NO.: 51772-016WO2

[0226] 2002) and Bowles et al. (J. Virol. 77:423-432, 2003), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

[0227] The sequences of naturally existing capsid proteins associated with AAV serotypes are known in the art and include those disclosed herein as VP1 , VP2, or VP3 of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhl 0, and AAVrh74.

[0228] Table 1 illustrates several, non-limiting examples of naturally occurring AAV capsid proteins.

[0229] Table 1. Capsid Proteins of AAV Serotypes PATENT

[0230] ATTORNEY DOCKET NO.: 51772-016WO2 PATENT

[0231] ATTORNEY DOCKET NO.: 51772-016WO2 PATENT

[0232] ATTORNEY DOCKET NO.: 51772-016WO2 PATENT

[0233] ATTORNEY DOCKET NO.: 51772-016WO2 PATENT

[0234] ATTORNEY DOCKET NO.: 51772-016WO2 PATENT

[0235] ATTORNEY DOCKET NO.: 51772-016WO2 PATENT

[0236] ATTORNEY DOCKET NO.: 51772-016WO2 rAAV virions useful in conjunction with the compositions and methods described herein include those derived from a variety of AAV serotypes including AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV13, AAVrhI O, AAVrh8, and AAVrh74, among others. Construction and use of AAV vectors and AAV proteins of different serotypes are described, for example, in Chao et al., Mol. Ther. 2:619-623, 2000), Davidson et al. {Proc. Natl. Acad. Sci. USA 97:3428-3432, 2000), Xiao et al. {J. Virol. 72: 2224-2232, 1998), Halbert et al. {J. Virol. 74: 1524-1532, 2000); Halbert et al. {J. Virol. 75: 6615-6624, 2001 ), and Auricchio et al. {Hum. Molec. Genet. 10: 3075-3081 , 2001 ), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

[0237] The nucleic acids and vectors described herein may also comprise a promoter sequence to initiate expression of a gene (e.g., a transgene). In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter such as, e.g., a tetracyclineinducible promoter that induces gene expression in the presence of tetracycline or doxycycline. In some embodiments, the promoter is a naturally occurring promoter, such that the promoter is found in an organism (e.g., a target organism intended to receive delivery or administration of one or more polynucleotides described herein). In some embodiments, the promoter is naturally occurring such that it is native to a gene comprising the transgene of interest. In further embodiments, the promoter is a synthetic promoter. Additional exemplary promoters that are useful for the expression of a transgene include, but are not limited to, a respiratory syncytial virus (RSV) promoter, a cytomegalovirus (CMV) promoter, an elongation factor 1 a (EF1 a) promoter, a simian virus 40 (SV40) promoter, a muscle creatine kinase (MCK) promoter, a desmin promoter, a myosin light-chain (MLC) promoter, a cardiac troponin T (cTnT) promoter, a synapsin (Syn) promoter, a chicken p-actin promoter with CMV enhancer elements (CB7), a tetracycline-controlled transactivator protein (tTA) promoter, an upstream activating sequence (UAS) promoter, a homeobox protein 9 (HB9) promoter, a CD68 molecule (CD68) promoter, a platelet- PATENT

[0238] ATTORNEY DOCKET NO.: 51772-016WO2 derived growth factor beta chain promoter, a reverse tetracycline-controlled transactivator protein (rTA) promoter, a U1 promoter, a U6 promoter, and a U7 promoter, or variations thereof.

[0239] In some embodiments, the AAV may include muscle specific regulatory elements such as a muscle specific promoter and / or a muscle specific enhancer. In some embodiments, the AAV may include a muscle specific promoter. Tissue-specific regulatory elements are known in the art. Non limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al, 1987, Genes Dev. 1 : 268-277) and muscle specific promoters (Skopenkova, et al, 2021 . Acta Naturae 13:47-58). In some embodiments, the muscle specific promoter is a phosphoglycerate kinase (PGK) promoter, a desmin promoter, a muscle creatine kinase promoter, a myosin light chain promoter, a myosin heavy chain promoter, a cardiac troponin C promoter, a troponin I promoter, a myoD gene family promoter, an actin alpha promoter, an actin beta promoter, an actin gamma promoter, or a promoter within intron 1 of ocular paired like homeodomain 3, a cytomegalovirus promoter, or a chicken-p-actin promoter. In some embodiments, the AAV may include a muscle specific enhancer. In some embodiments, the muscle specific enhancer is a muscle creatine kinase enhancer, a distal regulatory element (DRE) enhancer, a smooth muscle myosin heavy chain (SMHC) enhancer, a dystrophin intron 1 enhancer, a MyoD enhancer, or a myocyte enhancer factor 2 (MEF2) enhancer.

[0240] Also useful in conjunction with the compositions and methods described herein are pseudotyped rAAV vectors. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV2) pseudotyped with a capsid gene derived from a serotype other than the given serotype (e.g., AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV13, AAVrhI O, AAVrh8, and AAVrh74, among others). For example, a representative pseudotyped vector is an AAV2 vector encoding a therapeutic protein pseudotyped with a capsid gene derived from AAV serotype 8 or AAV serotype 9. In some embodiments, the pseudotyped AAV has the ITRs of one AAV serotype (e.g., AAV2) and the VP1 , VP2, and / or VP3 capsid proteins from a different AAV serotype (e.g., AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhI O, AAVrh8, or AAVrh74). Techniques involving the construction and use of pseudotyped rAAV virions are known in the art and are described, for example, in Duan et al., J. Virol. 75:7662-7671 (2001 ); Halbert et al. (J. Virol. 74:1524-1532, 2000); Zolotukhin et al. (Methods. 28:158-167, 2002); and Auricchio et al. (Hum. Molec. Genet. 10:3075-3081 , 2001 ).

[0241] In some embodiments, the AAV comprises a capsid disclosed, e.g., in WO 2017 / 218842, the disclosure of which is incorporated herein by reference. In some embodiments, the AAV comprises a capsid protein disclosed in Lin et al. (Mol Brain. 13:138, 2020), the disclosure of which is incorporated herein by reference. In some embodiments, the AAV comprises an AAV2-retro or an AAV9-retro capsid protein. In some embodiments, the AAV comprises a capsid protein that is conjugated to a ligand or an aptamer.

[0242] AAV virions that have mutations within the virion capsid may be used to infect particular cell types more effectively than non-mutated capsid virions. For example, suitable AAV mutants may have ligand insertion mutations for the facilitation of targeting AAV to specific cell types. The construction and characterization of AAV capsid mutants including insertion mutants, alanine screening mutants, and epitope tag mutants is described in Wu et al. (J. Virol. 74:8635-45, 2000). Other rAAV virions that can be used in methods of the invention include those capsid hybrids that are generated by molecular breeding PATENT

[0243] ATTORNEY DOCKET NO.: 51772-016WO2 of viruses as well as by exon shuffling. See, e.g., Soong et al. {Nat. Genet. 25:436-439, 2000) and Kolman and Stemmer Nat. Biotechnol. 19:423-428, 2001 ).

[0244] C. Additional Methods of Delivering a Transgene to a Host Cell - Transfection Techniques

[0245] Techniques that can be used to introduce a transgene (such as a floxed transgene encoding a therapeutic protein or a floxed transgene expression cassette comprising a transgene encoding a therapeutic protein) as described herein into a host cell (e.g., a host cell in a subject having or at risk of a disease or condition (e.g., a human subject)) are known in the art. For example, electroporation can be used to permeabilize mammalian cells (e.g., human cells) by the application of an electrostatic potential to the cell of interest. Mammalian cells, such as human cells, subjected to an external electric field in this manner are subsequently predisposed to the uptake of exogenous nucleic acids. The electroporation of mammalian cells is described in detail, e.g., in Chu et al. {Nucleic Acids Res. 15: 1311 , 1987), the disclosure of which is incorporated herein by reference. A similar technique, NUCLEOFECTION™, utilizes an applied electric field in order to stimulate the uptake of exogenous polynucleotides into the nucleus of a eukaryotic cell. NUCLEOFECTION™ and protocols useful for performing this technique are described in detail, e.g., in Distler et al. {Exp. Dermatol. 14:315, 2005), as well as in US 2010 / 0317114, the disclosures of each of which are incorporated herein by reference.

[0246] Additional techniques useful for the transfection of target cells include the squeeze-poration methodology. This technique induces the rapid mechanical deformation of cells in order to stimulate the uptake of exogenous DNA through membranous pores that form in response to the applied stress. This technology is advantageous in that a vector is not required for delivery of nucleic acids into a cell, such as a human target cell. Squeeze-poration is described in detail, e.g., in Sharei et al. {J. Vis. Exp. 81 :e50980, 2013), the disclosure of which is incorporated herein by reference.

[0247] Lipofection represents another technique useful for transfection of target cells. This method involves the loading of nucleic acids into a liposome, which often presents cationic functional groups, such as quaternary or protonated amines, towards the liposome exterior. This promotes electrostatic interactions between the liposome and a cell due to the anionic nature of the cell membrane, which ultimately leads to uptake of the exogenous nucleic acids, for example, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex. Lipofection is described in detail, for example, in US Patent No. 7,442,386, the disclosure of which is herein incorporated by reference. Similar techniques that exploit ionic interactions with the cell membrane to provoke the uptake of foreign nucleic acids include contacting a cell with a cationic polymer-nucleic acid complex. Exemplary cationic molecules that associate with polynucleotides so as to impart a positive charge favorable for interaction with the cell membrane are activated dendrimers (described, e.g., in Dennig {Topics in Current Chemistry 228:227, 2003), the disclosure of which is incorporated herein by reference) and diethylaminoethyl (DEAE)-dextran, the use of which as a transfection agent is described in detail, for example, in Gulick et al. {Curr. Protoc. in Mol. Biol. 40:1:9.2:9.2.1 , 1997), the disclosure of which is incorporated herein by reference. Magnetic beads are another tool that can be used to transfect target cells in a mild and efficient manner, as this methodology utilizes an applied magnetic field in order to direct the uptake of PATENT

[0248] ATTORNEY DOCKET NO.: 51772-016WO2 nucleic acids. This technology is described in detail, for example, in US 2010 / 0227406, the disclosure of which is incorporated herein by reference.

[0249] Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is laserfection, a technique that involves exposing a cell to electromagnetic radiation of a particular wavelength in order to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. This technique is described in detail, e.g., in Rhodes et al. (Methods in Cell Biology 82:309, 2007), the disclosure of which is incorporated herein by reference. Laserfection is also called optical transfection, which is a technique that involves exposing a cell to electromagnetic radiation of a particular wavelength in order to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. The bioactivity of this technique is similar to, and in some cases found superior to, electroporation.

[0250] Impalefection is another technique that may be used to deliver genetic material to target cells. It relies on the use of nanomaterials, such as carbon nanofibers, carbon nanotubes, and nanowires. Needle-like nanostructures are synthesized perpendicular to the surface of a substrate. DNA containing the gene, intended for intracellular delivery, is attached to the nanostructure surface. A chip with arrays of these needles is then pressed against cells or tissue. Cells that are impaled by nanostructures may express the delivered gene(s). An example of this technique is described in Shalek et al., PNAS 107: 1870 (2010), the disclosure of which is incorporated herein by reference.

[0251] Magnetofection may also be used to deliver nucleic acids to target cells. The magnetofection principle is to associate nucleic acids with cationic magnetic nanoparticles. The magnetic nanoparticles are made of iron oxide, which is fully biodegradable, and coated with specific cationic proprietary molecules varying upon the applications. Their association with the gene vectors (DNA, siRNA, viral vector, etc.) is achieved by salt-induced colloidal aggregation and electrostatic interaction. The magnetic particles are then concentrated on the target cells by the influence of an external magnetic field generated by magnets. This technique is described in detail in Scherer et al., Gene Therapy 9:102 (2002), the disclosure of which is incorporated herein by reference.

[0252] Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is sonoporation, a technique that involves the use of sound (typically ultrasonic frequencies) for modifying the permeability of the cell plasma membrane permeabilize the cells and allow polynucleotides to penetrate the cell membrane. This technique is described in detail, e.g., in Rhodes et al., Methods in Cell Biology 82:309 (2007), the disclosure of which is incorporated herein by reference.

[0253] Microvesicles represent another potential vehicle that can be used to modify the genome of a target cell according to the methods described herein. For example, microvesicles that have been induced by the co-overexpression of the glycoprotein VSV-G with, e.g., a genome-modifying protein, such as a nuclease, can be used to efficiently deliver proteins into a cell that subsequently catalyze the sitespecific cleavage of an endogenous polynucleotide sequence so as to prepare the genome of the cell for the covalent incorporation of a polynucleotide of interest, such as a gene or regulatory sequence. The use of such vesicles, also referred to as Gesicles, for the genetic modification of eukaryotic cells is described in detail, e.g., in Quinn et al., Mol. Then 23: Supplement 1 , Abstract No. 122, 2015). PATENT

[0254] ATTORNEY DOCKET NO.: 51772-016WO2

[0255] D. Methods of Genetic Engineering of a Host Cell

[0256] In addition to the methods described herein, a host cell may be modified to comprise a transgene (such as a floxed transgene encoding a therapeutic protein or a floxed transgene expression cassette comprising a transgene encoding a therapeutic protein) via a targeted integration method or other suitable gene editing technologies. Such methods may include the use of a site-specific nuclease, a transposase, a transcription activator-like effector nuclease (TALEN), mega-nuclease, zinc finger nuclease, a Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-associated protein 9 (CRISPR / Cas9)-based approach, homologous recombination, prime editing, transposon mediated delivery, in which an exogenous polynucleotide (e.g., a polynucleotide comprising a transgene that encodes a protein of interest) may be integrated into the genome of a host cell.

[0257] (i) CRISPR

[0258] One of the tools for the integration of target transgenes (such as a floxed transgene encoding a therapeutic protein or a floxed transgene expression cassette comprising a transgene encoding a therapeutic protein) into the genome of a target cell is the CRISPR / Cas system, a system that originally evolved as an adaptive defense mechanism in bacteria and archaea against viral infection. The CRISPR / Cas system includes palindromic repeat sequences within plasmid DNA and an associated Cas9 nuclease. This ensemble of DNA and protein directs site specific DNA cleavage of a target sequence by first incorporating foreign DNA into CRISPR loci. Polynucleotides containing these foreign sequences and the repeat-spacer elements of the CRISPR locus are in turn transcribed in a host cell to create a guide RNA, which can subsequently anneal to a target sequence and localize the Cas9 nuclease to this site. In this manner, highly site-specific cas9-mediated DNA cleavage can be engendered in a foreign polynucleotide because the interaction that brings cas9 within close proximity of the target DNA molecule is governed by RNA:DNA hybridization. As a result, one can design a CRISPR / Cas system to cleave any target DNA molecule of interest. This technique has been exploited in order to edit eukaryotic genomes (Hwang et al., Nature Biotechnology 31 :227 (2013)) and can be used as an efficient means of site- specifically editing target cell genomes in order to cleave DNA prior to the incorporation of a gene encoding a target gene. The use of CRISPR / Cas to modulate gene expression has been described in, for example, US Patent No. 8,697,359, the disclosure of which is incorporated herein by reference as it pertains to the use of the CRISPR / Cas system for genome editing.

[0259] A host cell may be modified to comprise a polynucleotide that comprises a transgene using CRISPR technology. “CRISPR” is programmable technology that targets specific stretches of genetic code to edit DNA at precise locations. CRISPR technology may include CRISPR-Cas9. Cas9 is a nuclease that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence, allowing for the insertion of exogenous nucleic acids into a cell’s genome. For example, CRISPR-based gene editing techniques can be used to introduce into a host cell genome, a transgene that encodes a protein of interest.

[0260] Exemplary CRISPR systems include those that utilize a Cas9 nuclease. Cas9 nuclease, together with CRISPR sequences, form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes within organisms. CRISPR technology may include Class 1 CRISPR systems including type I PATENT

[0261] ATTORNEY DOCKET NO.: 51772-016WO2

[0262] (cas3), type III (cas10), and type IV and 12 subtypes. CRISPR technology may include Class 2 CRISPR systems including type II (cas9), type V (cas12), type VI (cas13), and 9 subtypes. In some embodiments, CRISPR technology may involve CRISPR-Cas design tools which are computer software platforms and bioinformatics tools used to facilitate the design of guide RNAs (gRNAs) for use with the CRISPR / Cas gene editing system. For example, CRISPR-Cas design tools may include: CRISPRon, CRISPRoff, Invitrogen TrueDesign Genome Editor, Breaking-Cas, Cas-OFFinder, CASTING, CRISPy, CCTop, CHOPCHOP, CRISPOR, sgRNA Designer, Synthego Design Tool, and the like. CRISPR technology may also be used as a diagnostic tool. For example, CRISPR-based diagnostics may be coupled to enzymatic processes, such as SHERLOCK-based Profiling of in vitro Transcription (SPRINT). SPRINT can be used to detect a variety of substances, such as metabolites in subject samples or contaminants in environmental samples, with high throughput or with portable point-of-care devices.

[0263] Without being bound by theory, the mechanism of action of certain CRISPR nucleases includes the step of forming an R-loop whereby the CRISPR nuclease induces the unwinding of a double-strand DNA target, thereby separating the strands in the region bound by the CRISPR nuclease. The guide RNA spacer then hybridizes to the target strand at the protospacer sequence. This displaces a non-target strand that is complementary to the target strand, which forms the single strand region of the R-loop.

[0264] In some embodiments, the CRISPR nuclease includes one or more nuclease activities, which then cut the DNA leaving various types of lesions. For example, the CRISPR nuclease may comprise a nuclease activity that cuts the non-target strand at a first location, and / or cuts the target strand at a second location. In some embodiments, a CRISPR nuclease can cut zero, one, or two strands of a target nucleic acid. In some embodiments, the CRISPR nuclease is a nickase, which cuts one strand of a target nucleic acid. In some embodiments, the CRISPR nuclease is catalytically dead, which cuts zero strands of a target nucleic acid.

[0265] In some embodiments, the CRISPR nuclease comprises any one of the amino acid sequences as set forth herein. In some embodiments the CRISPR nuclease comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences set forth herein.

[0266] Examples of CRISPR nucleases include, without limitation, Cas9 (e.g., catalytically dead Cas9 (dCas9) and nickase Cas9 (nCas9)), Cas12a / Cpf1 , Cas12b / C2c1 , Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, and Cas12j / Cas<t>, Cas1 , Cas1 B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Casi o, Cas10d, Csy1 , Csy2, Csy3, Csy4, Cse1 , Cse2, Cse3, Cse4, Cse5e, Csc1 , Csc2, Csa5, Csn1 , Csn2, Csm1 , Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1 , Cmr3, Cmr4, Cmr5, Cmr6, Csb1 , Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1 , Csx1 S, Csx11 , Csf1 , Csf2, CsO, Csf4, Csd1 , Csd2, Cst1 , Cst2, Csh1 , Csh2, Csa1 , Csa2, Csa3, Csa4, Csa5, Type II Cas effector proteins, Type V Cas effector proteins, Type VI Cas effector proteins, CARF, DinG, homologs thereof, or modified or engineered versions thereof.

[0267] In some embodiments, the CRISPR system comprises a gene editor. In some embodiments, the gene editor comprises a CRISPR nuclease. The CRISPR nuclease of the gene editor may be any PATENT

[0268] ATTORNEY DOCKET NO.: 51772-016WO2

[0269] CRISPR nuclease described herein. In some embodiments, the CRISPR nuclease of the gene editor is a nCas. In some embodiments, the CRISPR nuclease of the base editor is a dCas. In some embodiments, the gene editor of the disclosure further comprises an (nuclear localization signal) NLS domain.

[0270] In some embodiments, the gene editor is a fusion protein, and the components of the gene editor are domains of the fusion protein, optionally connected by a linker. In some embodiments, the gene editor is a multi-protein complex, and the components of the gene editor are provided as individual polypeptides. In some embodiments, the gene editor is a multi-protein complex, and one or more components of the gene editor are provided endogenously by the cell.

[0271] In some embodiments, the CRISPR system comprises a prime editor. In some embodiments, the prime editor comprises a CRISPR nuclease and a reverse transcriptase (RT) polypeptide. In some embodiments, the gRNA for use with the prime editor is a prime editing gRNA (PEgRNA). The CRISPR nuclease of the gene editor may be any CRISPR nuclease described herein. In some embodiments, the CRISPR nuclease of the gene editor is a nCas. In some embodiments, the CRISPR nuclease of the base editor is a dCas. In some embodiments, the prime editors further comprise a flap endonuclease polypeptide. In some embodiments, the flap endonuclease polypeptide of the prime editor is a FEN1 domain. In some embodiments, the prime editors further comprise an NLS sequence.

[0272] In some embodiments, the prime editor is a fusion protein, and the components of the prime editor are domains of the fusion protein, optionally connected by a linker. In some embodiments, the prime editor is a multi-protein complex, and the components of the prime editor are provided as individual polypeptides. In some embodiments, the prime editor is a multi-protein complex, and one or more components of the prime editor are provided endogenously by the cell.

[0273] (ii) Transposon System

[0274] In addition to the above, a variety of tools have been developed that can be used for the incorporation of a transgene, such as a floxed transgene encoding a therapeutic protein or a floxed transgene expression cassette comprising a transgene encoding a therapeutic protein, into a target cell, and particularly into a human cell. One such method that can be used for incorporating polynucleotides into target cells involves the use of transposons.

[0275] A host cell may be modified to comprise a polynucleotide that comprises a transgene using a transposon system. Transposons or transposable elements include a (short) nucleic acid sequence with terminal repeat sequences upstream and downstream thereof. Transposons are polynucleotides that encode transposase enzymes and contain a polynucleotide sequence (e.g., a polynucleotide sequence that comprises a transgene) flanked by excision sites at the 5’ and 3’ positions. Once a transposon has been delivered into a cell, expression of the transposase gene commences and results in active enzymes that cleave the polynucleotide from the transposon. This activity is mediated by the site-specific recognition of transposon excision sites by the transposase.

[0276] In some embodiments, these excision sites may be terminal repeats or inverted terminal repeats. Once excised from the transposon, the polynucleotide (e.g., the polynucleotide that comprises a transgene) can be integrated into the genome of a host cell by transposase-catalyzed cleavage of similar excision sites that exist within nuclear genome of the cell. This allows the polynucleotide to be inserted PATENT

[0277] ATTORNEY DOCKET NO.: 51772-016WO2 into the cleaved nuclear DNA at the excision sites, and subsequent ligation of the phosphodiester bonds that join the polynucleotide of interest to the DNA of the host cell genome completes the incorporation process. In some embodiments, the transposon may be a retrotransposon, such that the polynucleotide (e.g., a DNA sequence that comprises a transgene) is first transcribed to an RNA product and then reverse-transcribed to DNA before incorporation in the prokaryotic or eukaryotic cell genome. Exemplary transposon systems include the Frog Prince transposon, the Tol2 transposon, piggyBac transposon (described in detail in, e.g., WO 2010 / 085699), and the Sleeping Beauty transposon (described in detail in, e.g., US 2005 / 0112764), the disclosures of each of which are incorporated herein by reference as they pertain to transposons for use in gene delivery to a cell of interest.

[0278] (iii) Mega-nucleases

[0279] Additional genome editing techniques that can be used to incorporate polynucleotides encoding target transgenes into the genome of a target cell include the use of mega-nucleases such as ARCUS™ mega-nucleases that can be rationally designed so as to site-specifically cleave genomic DNA. The use of these enzymes for the incorporation of genes encoding target genes into the genome of a mammalian cell is advantageous in view of the defined structure-activity relationships that have been established for such enzymes. Single chain mega-nucleases can be modified at certain amino acid positions in order to create nucleases that selectively cleave DNA at desired locations, enabling the site-specific incorporation of a target transgene into the nuclear DNA of a target cell. These single-chain nucleases have been described extensively in, for example, US Patent Nos. 8,021 ,867 and US 8,445,251 , the disclosures of each of which are incorporated herein by reference as they pertain to compositions and methods for genome editing.

[0280] (iv) TALENs

[0281] Alternative methods for site-specifically cleaving genomic DNA prior to the incorporation of a transgene of interest in a target cell include the use of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Unlike the CRISPR / Cas system, these enzymes do not contain a guiding polynucleotide to localize to a specific target sequence. Target specificity is instead controlled by DNA binding domains within these enzymes. The use of ZFNs and TALENs in genome editing applications is described, e.g., in Urnov et al., Nature Reviews Genetics 11 :636 (2010); and in Joung et al., Nature Reviews Molecular Cell Biology 14:49 (2013), the disclosure of each of which are incorporated herein by reference as they pertain to compositions and methods for genome editing.

[0282] II. Cre-lox System

[0283] Described herein is the Cre-lox recombination system that can be used in conjunction with the compositions and methods of the disclosure for, e.g., modulating the expression of a previously delivered transgene of interest.

[0284] A. Cre recombinase and lox sites PATENT

[0285] ATTORNEY DOCKET NO.: 51772-016WO2

[0286] Cre recombinase is a DNA-binding small bacteriophage P1 -derived tyrosine recombinase enzyme. It uses a topoisomerase l-like mechanism to catalyze DNA recombination events between two specific DNA recognition sites, named lox sites, without requiring high-energy cofactors. The 34 base pairs (hereinafter "bp”) lox sites are composed of two 13 bp palindromic regions (recombinase binding elements) flanking an asymmetric 8 bp spacer sequence. The latter determines the orientation of the lox sites. When a DNA fragment is flanked by two lox sites in the same orientation, Cre excises it as a circular molecule, leaving a single lox site behind. Conversely, when lox sites are in opposing orientations, Cre reverses the flanked DNA. Moreover, the spacer sequence also defines the identify of a target site, as heterospecific mutants efficiently cross-interact with one another, but rarely with the wildtype lox site.

[0287] Cre recombinase is a 38.5 kDa protein with 343 amino acids that form two distinct domains. The enzyme Cre recombinase and lox sites originated from bacteriophage P1 . Cre or Cre recombinase is a tyrosine site-specific recombinase. In bacteriophage P1 , the lox sites are called loxP sites (locus of crossing over in phage P1 ). Lox sites are short DNA target sequences. The lox sites consist of a 34-bp sequence in which there is an 8-bp spacer region and two 13-bp inverted repeats. The two 13-bp inverted repeats are separated by the spacer region. This 34-bp target sequence is required for the site-specific recombination process in combination with the Cre protein which mediates the recombination by recognizing the lox sites. In some embodiments, the transgene expression cassette comprising the open reading frame of the gene (e.g., a therapeutic transgene encoding a therapeutic protein) and other functional components is flanked by the lox sites, thus generating a floxed transgene expression cassette. In some embodiments, the floxed transgene expression cassette is the one being genetically manipulated by the Cre-lox system. In some embodiments, the open reading frame of the gene (e.g., a therapeutic transgene encoding a therapeutic protein) is flanked by the lox sites, thus generating a floxed gene. In some embodiments, the floxed gene is the one being genetically manipulated by the Cre-lox system. In some embodiments, the functional component is a promoter, an enhancer, or a terminator, such as a pA signal. In some embodiments, the promoter is flanked by the lox sites, thus generating a “floxed” promoter. In some embodiments, the “floxed” promoter is the one being genetically manipulated by the Cre-lox system. In some embodiments, the enhancer is flanked by the lox sites, thus generating a “floxed” enhancer. In some embodiments, the “floxed” enhancer is the one being genetically manipulated by the Cre-lox system. In some embodiments, the terminator is flanked by the lox sites, thus generating a “floxed” terminator. In some embodiments, the “floxed” terminator is the one being genetically manipulated by the Cre-lox system. In some embodiments, the “floxed” terminator is a “floxed” pA signal.

[0288] B. Mechanism of Action

[0289] Cre recombinase is used in both prokaryotic and eukaryotic systems. Cre recombinase is used widely for site-specific recombination in yeast and mammalian cells and in this site-specific recombination process, crossing-over takes place at the lox sites. Cre is the phage-encoded protein that carries out this recombination between two lox sites by recognizing these DNA target sequences.

[0290] The Cre-lox system can be used to carry out deletions or excisions, insertions, translocations, and inversions, thus, allowing genes to be activated, deactivated, or replaced by another gene. The type PATENT

[0291] ATTORNEY DOCKET NO.: 51772-016WO2 of genetic modification carried out by the Cre-lox system such as deletion or excision, insertion, translocation, or inversion depends on the on the orientation and location of lox sites. A single Cre recombinase enzyme recognizes two directly repeated lox sites, then Cre excises the lox flanked (fioxed) DNA, which creates two types of DNA sequences, one with the circular, excised, and inactivated gene and one of the lox sites and the other one with the second lox site. The removal of the inactivated gene and one of the lox sites is followed by DNA ligation, thus resulting in a circular sequence. Gene excision is the most common genetic modification carried out by the Cre-lox system. For gene deletion or excision, the 2 lox sequences are placed with similar orientation on either side of a target DNA sequence. To accomplish induced gene expression or gene activation, a stop codon is fioxed with two identical lox sequences such that the Cre enzyme can remove the transcription stop codon and result in expression of the gene. To accomplish gene inversion, the target gene is fioxed between two lox sequences of opposite orientations such that the Cre enzyme can invert the sequence of the gene. To accomplish gene translocation, two identically oriented lox sites are located on two separate chromosomes (each with a different gene) such that the Cre protein can mediate the translocation or swapping of the genes between the two chromosomes. Cre recombinase can also be used for insertion of donor DNA sequences. To accomplish insertion of donor DNA, a donor DNA cassette that consists of a lox sequence and the donor sequence is required. The site for the cassette insertion is marked by a second lox sequence on the target chromosome. Insertion of the donor sequence into the target chromosome of the host is accomplished through Cre-lox recombination.

[0292] Using this system, DNA modification can be carried out selectively in the specific cell type desired, for example, by placing the Cre enzyme under the control of a cell type or tissue-specific promoter or enhancer such as a muscle specific promoter or enhancer. Exemplary muscle specific Cre promoters of the disclosure include ACTA1 (HSA), Ckmm (Mck), Myf5, Myh6 (aMHC), and Talgn (SM22a). Tissue-specific regulatory elements are known in the art. Non limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al, 1987. Genes Dev. 1 : 268-277) and muscle-specific promoters (Skopenkova, et al, 2021 . Acta Naturae 13:47-58). In some embodiments, the muscle specific promoter is a phosphoglycerate kinase (PGK) promoter, a desmin promoter, a muscle creatine kinase promoter, a myosin light chain promoter, a myosin heavy chain promoter, a cardiac troponin C promoter, a troponin I promoter, a myoD gene family promoter, an actin alpha promoter, an actin beta promoter, an actin gamma promoter, or a promoter within intron 1 of ocular paired like homeodomain 3, a cytomegalovirus promoter, or a chicken-p-actin promoter. In some embodiments, the Cre enzyme may be placed under the control of a muscle-specific enhancer. In some embodiments, the muscle-specific enhancer is a muscle creatine kinase enhancer, a distal regulatory element (DRE) enhancer, a smooth muscle myosin heavy chain (SMHC) enhancer, a dystrophin intron 1 enhancer, a MyoD enhancer, or a myocyte enhancer factor 2 (MEF2) enhancer.

[0293] The Cre-lox system can also be used for temporal control of gene expression by providing the Cre after a certain period after the administration of the fioxed transgene encoding the therapeutic protein or the fioxed transgene expression cassette comprising a transgene encoding a therapeutic protein. This enables the therapeutic protein to be expressed for a certain amount of time before its expression can be terminated. In some embodiments, the Cre can be administered to the muscle via intramuscular (IM) PATENT

[0294] ATTORNEY DOCKET NO.: 51772-016WO2 administration and could reach the transduced cells in a local manner while limiting system-wide exposure to the recombinase. Thus, this system enables genetic manipulation with high efficiency in a tissue-specific or time-specific manner. The Cre-lox system can also function in an inducible manner. This also adds to its tissue-specificity and temporal-specificity. The Cre-lox system, when inducible is controlled by cell-specific regulatory elements (promoters and enhancers, such as a muscle specific promoter or enhancer) and it can be made temporally inducible by an external inducer or trigger such as tamoxifen (tarn), tetracycline (tet), or antibiotic TMP. When the external trigger is tarn, the system is called a tam-inducible system and when the external tugger is doxycycline or dox (a tetracycline derivative), the system is called a dox-inducible system or tet-system. In such cases, administration of tarn or dox can manipulate the activity of the Cre-lox system.

[0295] In some embodiments, the Cre recombinase is applied in saturation to eliminate transgene expression. In some embodiments, the Cre recombinase is administered to the subject in a transient manner. In some embodiments, the Cre recombinase is expressed in the subject in a transient manner. In some embodiments, the Cre recombinase is expressed in a transient manner in the cells of the subject harboring the floxed transgene or the floxed transgene expression cassette comprising a transgene encoding a therapeutic protein. In some embodiments, the Cre can be administered to the muscle via IM administration and could reach the transduced cells in a local manner while limiting system-wide exposure to the recombinase. In some embodiments, the floxed transgene or the floxed transgene expression cassette comprising a transgene encoding a therapeutic protein is expressed in the liver and the Cre is administered to reach the liver.

[0296] Exemplary Cre recombinase proteins include those having the amino acid sequences of SEQ ID NOs: 17-19. SEQ ID NO: 17 is wild-type Cre recombinase protein. SEQ ID NO: 17 is shown below:

[0297] MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRK WFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERA KQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLV STAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGA KDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLEDGD

[0298] SEQ ID NO: 18 is a Cre recombinase variant (truncated Cre recombinase; residues 19-343; sCre). SEQ ID NO: 18 is shown below:

[0299] SDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQAR GLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLME NSDRCQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSAR VGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED

[0300] SEQ ID NO: 19 is a Cre recombinase variant (DD-Cre). SEQ ID NO: 19 is shown below:

[0301] MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHTWESIGRPLPGRKNIILSSQP STDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESV PATENT

[0302] ATTORNEY DOCKET NO.: 51772-016WO2

[0303] FSEFHDADAQNSHSYCFEILERRGAPKKKRKVSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFS EHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPR PSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLLRIAEI ARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVA APSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTN VNIVMNYIRNLDSETGAMVRLLEDGD

[0304] An exemplary nucleic acid sequence encoding wild-type Cre recombinase is shown below (SEQ ID NO: 20):

[0305] TGCGCAGCTGGACGTAAACTCCTCTTCAGACCTAATAACTTCGTATAGCATACATTATAC

[0306] GAAGTTATATTAAGGGTTATTGAATATGATCAATTTACCTGTAAATCCATACAGTTCAAT ACCTTAGCAGGTCAAATAGTGACCACTTGATCATTTGATCAAGGTTGCGCTACGTAAAAT CTGTGAAAAATTGGCGGTGTTAGTCCTACAGATTTCGCGTACCACTTAGCACCACCAATC AATCAGAGGTGAAAAATGGGATATTCAACTGCTAAAGTGTCCACTCATCTTGAGCTTGAG AAAAACCGTGGTTACTGGCGGGCAAAAGGGTTTGATCGTGATAGTTGCCAACTGTCATTA TCGCGCGGTGAAGAGAAAATAGAACGCACGCGCGGTCGCTGGCGTTTCTATGACGAGAAC CATAAACAGGTAAAGGCAGAGCCGATCCTGTACACTTTACTTAAAACCATTATCTGAGTG TTAAATGTCCAATTTACTGACCGTACACCAAAATTTGCCTGCATTACCGGTCGATGCAAC GAGTGATGAGGTTCGCAAGAACCTGATGGACATGTTCAGGGATCGCCAGGCGTTTTCTGA GCATACCTGGAAAATGCTTCTGTCCGTTTGCCGGTCGTGGGCGGCATGGTGCAAGTTGAA TAACCGGAAATGGTTTCCCGCAGAACCTGAAGATGTTCGCGATTATCTTCTATATCTTCA GGCGCGCGGTCTGGCAGTAAAAACTATCCAGCAACATTTGGGCCAGCTAAACATGCTTCA TCGTCGGTCCGGGCTGCCACGACCAAGTGACAGCAATGCTGTTTCACTGGTTATGCGGCG GATCCGAAAAGAAAACGTTGATGCCGGTGAACGTGCAAAACAGGCTCTAGCGTTCGAACG CACTGATTTCGACCAGGTTCGTTCACTCATGGAAAATAGCGATCGCTGCCAGGATATACG TAATCTGG C ATTTCTG G GG ATTG CTTATAAC ACCCTGTTACGTATAG CCG AAATTG CC AG GATCAGGGTTAAAGATATCTCACGTACTGACGGTGGGAGAATGTTAATCCATATTGGCAG AACGAAAACGCTGGTTAGCACCGCAGGTGTAGAGAAGGCACTTAGCCTGGGGGTAACTAA ACTGGTCGAGCGATGGATTTCCGTCTCTGGTGTAGCTGATGATCCGAATAACTACCTGTT TTGCCGGGTCAGAAAAAATGGTGTTGCCGCGCCATCTGCCACCAGCCAGCTATCAACTCG CGCCCTGGAAGGGATTTTTGAAGCAACTCATCGATTGATTTACGGCGCTAAGGATGACTC TGGTCAGAGATACCTGGCCTGGTCTGGACACAGTGCCCGTGTCGGAGCCGCGCGAGATAT GGCCCGCGCTGGAGTTTCAATACCGGAGATCATGCAAGCTGGTGGCTGGACCAATGTAAA TATTGTCATGAACTATATCCGTAACCTGGATAGTGAAACAGGGGCAATGGTGCGCCTGCT GGAAGATGGCGATTAGCCATTAACGCGTAAATGATTGCTATAATTAGTTGATA

[0307] Exemplary lox sequences that may be used in conjunction with the compositions and methods described herein include those having the nucleic acid sequences of SEQ ID NOs: 21 -96 and are shown in Table 2 below. Specifically shown are the top and the bottom (reverse complementary) strand PATENT

[0308] ATTORNEY DOCKET NO.: 51772-016WO2 sequences for exemplary lox sites of the disclosure. All strands shown below are recited in the 5’ to 3’ direction.

[0309] Table 2. Exemplary lox sequences of the disclosure that can be used for Cre-lox recombination. PATENT

[0310] ATTORNEY DOCKET NO.: 51772-016WO2 PATENT

[0311] ATTORNEY DOCKET NO.: 51772-016WO2

[0312] *WT: wild-type

[0313] C. Delivery of Cre recombinase

[0314] Using the compositions and methods described herein, Cre recombinase can be delivered into a host cell to enable Cre-lox recombination. Cre recombinase can be delivered into a host cell using any of the Cre delivery techniques described herein, which can be classified into two groups: viral mode of delivery and non-viral mode of delivery.

[0315] (i) Viral Delivery of Cre recombinase

[0316] In some embodiments, Cre recombinase is delivered using a viral mode of delivery. In some embodiments, Cre recombinase is delivered using a viral vector in section 1(B). Examples of viral vectors that may be used in conjunction with the compositions and methods described herein are adeno- associated virus (AAV), retrovirus, adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses that may be used in conjunction with the compositions and methods described herein include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). Other examples include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses. Other examples of vectors are described, for example, in US Patent No. 5,801 ,030, the disclosure of which is incorporated herein by reference as it pertains to viral vectors for use in gene therapy.

[0317] In some embodiments, Cre recombinase is delivered using a viral vector such as an AAV. In some embodiments, the viral vector is a recombinant AAV (rAAV). In some embodiments, the AAV is an AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV13, AAVrh74, AAVrh8, or AAVrhI O serotype. In some embodiments, the AAV is a pseudotyped AAV, such as an AAV2 / 8 or AAV2 / 9. PATENT

[0318] ATTORNEY DOCKET NO.: 51772-016WO2

[0319] (ii) Non-viral Delivery of Cre recombinase

[0320] In some embodiments, Cre recombinase is delivered using a non-viral mode of delivery. Non-viral modes of delivery provide several advantages such as wide raw materials, flexible chemical composition, easily modulated topology, high DNA loading, and safety. Non-viral modes of delivery for Cre recombinase include peptide-based delivery systems, lipid-based delivery systems (e.g., lipid nanoparticles (LNPs)), inorganic delivery systems, polymeric delivery systems, exosomes, polysaccharide macromolecules, various transfection techniques (e.g., electroporation), and other modes of delivery using which an exogenous polynucleotide (e.g., a polynucleotide comprising a transgene that encodes a protein of interest such as Cre recombinase) may be integrated into the genome of a host cell such as using site-specific nucleases, transposases, transcription activator-like effector nucleases (TALENs), mega-nucleases, zinc finger nucleases, Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-associated protein 9 (CRISPR / Cas9)-based approaches, homologous recombination, prime editing, and transposon mediated delivery, among others.

[0321] In some embodiments, Cre recombinase is delivered using a peptide-based delivery system such as a cell-penetrating peptide (CPP). CPPs can facilitate the cellular intake and uptake of molecules and result in efficient delivery. In some embodiments, Cre recombinase is delivered using a lipid-based delivery system such as LNPs, liposomes, liposome-based nanoparticles, and cationic lipids, among others. In some embodiments, Cre recombinase is delivered using one or more LNPs, such as one or more of the LNP systems described in section 11(D). In some embodiments, a Cre recombinase may be formulated in an LNP, thus, generating an LNP formulated Cre (LNP-Cre) as described herein. In some embodiments, Cre recombinase is delivered using an inorganic delivery system. Inorganic delivery systems can be classified into several categories: quantum dots, carbon nanotubes, black phosphorus, graphene oxide, mesoporous silica nanoparticles, and gold nanoparticles. Gold nanoparticles can penetrate cell membranes and since gold is generally well tolerated by the human body, it can be easily conjugated with DNA. Inorganic delivery systems are stable, biocompatible, have sufficient surface areas with adjustable shapes, and possess large loading capacities. In some embodiments, Cre recombinase is delivered using a polymeric delivery system such as boronic dendrimer, nano-clew, Cas9 micelles, polyethylenimine complex, cationic polymers, polymer hydrogels, and polymeric nanocapsules. Polymeric delivery systems are polymeric formulations that can transport and deliver a therapeutic agent into a cell or body. They are safe, efficient, and can stably control the rate, time, and place of drug release. In some embodiments, Cre recombinase is delivered using exosomes. In some embodiments, Cre recombinase is delivered using polysaccharide macromolecules such as chitosan (CS), cyclodextrin (CD) and dextran. In some embodiments, Cre recombinase is delivered using a transfection technique in section 1(C). In some embodiments, a host cell may be modified to comprise a transgene such as Cre recombinase using any of the techniques in section 1(D).

[0322] D. Lipid Nanoparticles (LNPs)

[0323] Featured below are LNPs that can be used to deliver a Cre recombinase described herein. All these LNPs, modifications thereof, and pharmaceutical compositions thereof are useful for the methods described herein such as the methods of treatment described herein. A Cre recombinase may be PATENT

[0324] ATTORNEY DOCKET NO.: 51772-016WO2 formulated in an LNP, thus, generating, an LNP formulated Cre (LNP-Cre) described herein, which can be useful for the methods described herein.

[0325] The term “lipid nanoparticle” or “LNP” refers to a transfer or delivery vehicle including one or more lipids {e.g., cationic lipids, non-cationic lipids, and polyethylene glycol (PEG)-modified lipids). LNPs are nanoparticles composed of lipids. LNPs are generally spherical and have an average diameter ranging from 10 to 1000 nm. Examples of suitable lipids include, for example, the phosphatidyl compounds {e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides). Examples of suitable lipids also include, for example, triglycerides, diglycerides, monoglycerides, fatty acids, steroids, and waxes. LNPs may contain a cationic lipid, or a lipid species with a net positive charge at a selected pH {e.g., physiological pH), to encapsulate and / or enhance the delivery of a therapeutic product into the target cells. LNPs can deliver a variety of therapeutic agents such as small molecules, mRNA, mRNA vaccines, mRNA therapeutics, proteins such as Cre recombinase, nucieoside-modified messenger RNA (modRNA), nucleic acids, and small interfering RNA (siRNA) drugs, among others. In some embodiments, a Cre recombinase of the present disclosure is formulated in an LNP. Exemplary LNPs are formulated to deliver Cre recombinase to one or more target cells. LNPs may comprise an ionizable lipid {e.g., an ionizable amino lipid), a sterol or other structural lipid, a non-cationic helper lipid or phospholipid, and a PEG-modified lipid. Emulsifiers or surfactants stabilize lipid cores. Emulsifiers are used to stabilize lipid dispersion and they prevent particle agglomeration.

[0326] In some embodiments, a payload is formulated as a solid LNP (SLN), which can be spherical with an average diameter between 10 to 1000 nm and has only one phospholipid layer. SLNs possess a solid lipid core matrix that can solubilize lipophilic molecules and can be stabilized with surfactants and / or emulsifiers. A payload such as Cre recombinase can be embedded in the interior of an SLN. SLNs may have targeting compounds bound to their exterior surface such as antibodies, cell-targeting peptides, and drug molecules, among others. The relative amounts of the lipids and the Cre recombinase in an LNP composition of the present disclosure can be optimized according to considerations of efficacy and tolerability.

[0327] LNPs are carriers that provide a biocompatible and biodegradable delivery system for a payload such as a Cre recombinase as described herein. Nanostructured lipid carriers (NLCs) are modified SLNs that retain the characteristics of the SLN, improve drug stability and loading capacity, and prevent drug leakage. Polymer nanoparticles (PNPs) are a key component of drug delivery. These nanoparticles can effectively direct drug delivery to specific targets and improve drug stability and controlled drug release. Lipid-polymer nanoparticles (PLNs), a new type of carrier that combines liposomes and polymers, may also be employed. These nanoparticles possess the complementary advantages of PNPs and liposomes. A PLN is composed of a core-shell structure; the polymer core provides a stable structure, and the phospholipid shell offers good biocompatibility. As such, the two components increase the drug encapsulation efficiency rate, facilitate surface modification, and prevent leakage of water-soluble drugs.

[0328] Lipid components of LNPs, such as phospholipids (for example, phosphatidylcholine and phosphatidylethanoiamine), cholesterol, or polyethylene glycol (PEG)-functlonailzed lipids (PEG-ilpids) can help improve LNP properties, such as particle stability, delivery efficacy, tolerability, and PATENT

[0329] ATTORNEY DOCKET NO.: 51772-016WO2 biodistribution. Phosphatidylcholine derivatives can help destabilize endosomal membranes and facilitate endosomai escape of LNPs. Molecular shape and configuration of cholesterol derivatives can impact LNP delivery efficacy and biodistribution, for example, some cholesterol derivatives can affect the selectivity for the type of liver ceil. PEG-iipids enable targeted delivery through conjugation of specific ligands to LNPs and help prevent clearance by renal filtration. Organ selectivity can vary based on the relative proportions of the lipid components and administration routes. Intravenous administration may lead to accumulation of LNPs in the liver and lymph nodes. Biodistribution of LNPs is affected by the chemical composition, proportions of the individual components, shape, size, molecular geometry, and surface properties of the LNP. LNPs can be used to deliver therapeutic products intramuscularly, and LNPs help protect the therapeutic products from degradation before cellular uptake and endosomal escape into their cytoplasmic target compartment. In some embodiments, an LNP-Cre of the present disclosure is administered intramuscularly. In some embodiments, an LNP-Cre of the present disclosure is administered intravenously. In some embodiments, an LNP-Cre of the present disclosure is administered to reach the liver. In some embodiments, a Cre recombinase or a nucleic acid encoding Cre recombinase of the present disclosure is administered intramuscularly using any other Cre delivery technique described herein. In some embodiments, a Cre recombinase or a nucleic acid encoding Cre recombinase of the present disclosure is administered intravenously using any other Cre delivery technique described herein. In some embodiments, a Cre recombinase or a nucleic acid encoding Cre recombinase of the present disclosure is administered to reach the liver using any other Cre delivery technique described herein.

[0330] LNPs, in some embodiments, include one or more ionic lipids, such as non-cationic lipids (e.g., neutral, or anionic, or zwitterionic lipids); one or more conjugated lipids (such as PEG-conjugated lipids or lipids conjugated to polymers described in Table 5 of International Patent Publication No.

[0331] WO2019 / 217941 ; incorporated herein by reference in its entirety); and / or one or more sterols (e.g., cholesterol). Lipids that can be used in nanoparticle formations (e.g., LNPs) include, for example those described in Table 4 of International Patent Publication No. WO2019 / 217941 , which is incorporated by reference — e.g., a lipid-containing nanoparticle can include one or more of the lipids in Table 4 of International Patent Publication No. WO2019 / 217941 . LNPs can include additional elements, such as polymers, such as the polymers described in Table 5 of International Patent Publication No.

[0332] WO2019 / 217941 , incorporated by reference. In some embodiments, conjugated lipids, when present, can include one or more of PEG-diacylglycerol (DAG) (such as 1 -(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG- ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-1 -0-(w- methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, and N- (carbonyl-methoxypoly ethylene glycol 2000)-1 ,2-distearoyl-sn- glycero-3-phosphoethanolamine sodium salt, among others. In some embodiments, sterols that can be incorporated into LNPs include one or more of cholesterol or cholesterol derivatives, such as those in W02009 / 127060 or US2010 / 0130588, which are incorporated by reference.

[0333] In some embodiments, a composition described herein (e.g., a Cre recombinase) is provided in an LNP that includes an ionizable lipid. In some embodiments, an ionizable lipid may be a cationic lipid, PATENT

[0334] ATTORNEY DOCKET NO.: 51772-016WO2 an ionizable cationic lipid, e.g., a cationic lipid that can exist in a positively charged or neutral form depending on pH, or an amine-containing lipid that can be readily protonated. In some embodiments, the cationic lipid is a lipid capable of being positively charged, e.g., under physiological conditions. Exemplary cationic lipids include one or more amine group(s) which bear the positive charge. In some embodiments, the lipid particle includes a cationic lipid in formulation with one or more of neutral lipids, ionizable amine- containing lipids, biodegradable alkyne lipids, steroids, phospholipids including polyunsaturated lipids, structural lipids (e.g., sterols), PEG, cholesterol, and polymer conjugated lipids. In some embodiments, the cationic lipid may be an ionizable cationic lipid. In some embodiments, the Ore recombinase may be encapsulated in an LNP, e.g., an LNP including a cationic lipid. In some embodiments, the LNP may include a targeting moiety, e.g., coated with a targeting agent. In some embodiments, the LNP formulation is biodegradable.

[0335] In some embodiments, the LNP can include a PEG or a conjugated lipid molecule. Generally, these are used to inhibit aggregation of LNPs and / or provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, for example, a (methoxy polyethylene glycol)-conjugated lipid.

[0336] In some embodiments, one or more additional compounds can also be included. Those compounds can be administered separately, or the additional compounds can be included in the LNPs of the disclosure. In other words, the LNPs can contain other compounds in addition to the Cre recombinase. Without limitations, other additional compounds can be selected from the group consisting of small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives thereof, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, an extract made from biological materials, or any combinations thereof.

[0337] An LNP may optionally include one or more coatings. In some embodiments, an LNP may be formulated in a capsule, film, or tablet having a coating. A capsule, film, or tablet including a composition described herein may have any useful size, tensile strength, hardness, or density.

[0338] In some embodiments, the LNP formulations can be engineered to alter the surface properties of particles so that the LNPs can penetrate the mucosal barrier. The LNP engineered to penetrate mucus can comprise a polymeric material ( / .e., a polymeric core) and / or a polymer-vitamin conjugate and / or a triblock co-polymer. The polymeric material can include, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes), polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. LNPs engineered to penetrate mucus can also include surface altering agents such as, but not limited to, polynucleotides, anionic proteins {e.g., bovine serum albumin), surfactants {e.g., cationic surfactants such as for example dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives {e.g., cyclodextrin), nucleic acids, polymers {e.g., heparin, polyethylene glycol and poloxamer), mucolytic agents {e.g., N-acetylcysteine, mugwort, bromelain, papain, clerodendrum, acetylcysteine, bromhexine, carbocisteine, eprazinone, PATENT

[0339] ATTORNEY DOCKET NO.: 51772-016WO2 mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin p4 dornase alfa, neltenexine, erdosteine), and various DNases including rhDNase.

[0340] III. Pharmaceutical Compositions

[0341] The nucleic acids and vectors described herein may be formulated into various compositions (e.g., a pharmaceutical composition) for administration to a subject in a biologically compatible form suitable for administration in vivo. For example, the agents described herein may be administered in a suitable diluent, carrier, stabilizer, or excipient, and may further contain a preservative, e.g., to prevent the growth of microorganisms. Conventional procedures and ingredients for the selection and preparation of suitable compositions are described, for example, in Remington, J.P. The Science and Practice of Pharmacy, Easton, PA. Mack Publishers, 2012, 22nd ed. and in The United States Pharmacopeial Convention, The National Formulary, United States Pharmacopeial, 2015, USP 38 NF 33.

[0342] Mixtures of agents described herein may be prepared in water suitably mixed with one or more excipients, carriers, or diluents. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (described in US 5,466,468, the disclosure of which is incorporated herein by reference). In any case the formulation may be sterile and may be fluid to the extent that easy syringability exists. Formulations may be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0343] For example, a solution containing a pharmaceutical composition described herein may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intramuscular, intravenous, subcutaneous, and intraperitoneal administration.

[0344] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals (e.g., non-human mammals). Modification of pharmaceutical compositions suitable for administration to humans to render the compositions suitable for administration to various PATENT

[0345] ATTORNEY DOCKET NO.: 51772-016WO2 animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and / or other primates and mammals.

[0346] Compositions containing an agent such as a nucleic acid or a Cre recombinase described herein may include a delivery vehicle for administration to a subject or one or more cells thereof. Exemplary delivery vehicles for a Cre recombinase described herein include, but are not limited to, lipid-based carriers (e.g., lipid nanoparticle formulations such as LNP formulated Cre (LNP-Cre)), and suitable polymers.

[0347] Compositions containing an agent such as a nucleic acid or vector described herein may further include a second agent (e.g., a nucleic acid molecule to be expressed within a cell, a polypeptide, or a drug). For example, a second agent may be a blood pressure medication, steroid, an analgesic, or an immunosuppressive agent.

[0348] IV. Methods of Treatment

[0349] Any of the compositions described herein such as the nucleic acid molecules (e.g., polynucleotides comprising transgenes such as a floxed transgene or polynucleotides comprising floxed transgene expression cassettes comprising a transgene encoding a therapeutic protein), vectors and AAV vectors (e.g., rAAV vectors), Cre recombinase (such as LNP-Cre, or a Cre recombinase or a nucleic acid encoding Cre recombinase administered using any other Cre delivery technique described herein), and any pharmaceutical compositions thereof, can be used in a method of treatment for a disease or condition in a subject in need thereof (e.g., a human subject). In some embodiments, a method of treatment may be prophylactic treatment for a subject at risk of a disease or condition. In other embodiments, a method of treatment may reduce, reverse, ameliorate, stabilize, or improve a disease state or condition in a subject. In other embodiments, the methods may be used to alleviate, ameliorate, reduce, or reverse one or more clinical manifestations of a disease or condition.

[0350] In some embodiments, the method of treatment is directed to treating a metabolic disorder, a blood disorder, a cardiovascular disorder, a neurological disorder, an ocular or ophthalmological disorder, a reproductive disorder, an infectious disease, an autoimmune or immunological disorder, or a type of cancer. Exemplary diseases and target genes (e.g., suitable transgenes) that may benefit from the compositions and methods described herein are summarized in Table 3 below.

[0351] Table 3. Exemplary Diseases and Target Genes PATENT

[0352] ATTORNEY DOCKET NO.: 51772-016WO2 PATENT

[0353] ATTORNEY DOCKET NO.: 51772-016WO2 PATENT

[0354] ATTORNEY DOCKET NO.: 51772-016WO2 PATENT

[0355] ATTORNEY DOCKET NO.: 51772-016WO2 PATENT

[0356] ATTORNEY DOCKET NO.: 51772-016WO2 PATENT

[0357] ATTORNEY DOCKET NO.: 51772-016WO2 PATENT

[0358] ATTORNEY DOCKET NO.: 51772-016WO2 PATENT

[0359] ATTORNEY DOCKET NO.: 51772-016WO2 PATENT

[0360] ATTORNEY DOCKET NO.: 51772-016WO2 PATENT

[0361] ATTORNEY DOCKET NO.: 51772-016WO2 PATENT

[0362] ATTORNEY DOCKET NO.: 51772-016WO2 PATENT

[0363] ATTORNEY DOCKET NO.: 51772-016WO2 PATENT

[0364] ATTORNEY DOCKET NO.: 51772-016WO2 PATENT

[0365] ATTORNEY DOCKET NO.: 51772-016WO2

[0366] A. Heterologous Expression of a Transgene as a Method of Treatment

[0367] In some embodiments, a subject is administered an effective amount of a polynucleotide comprising a transgene (such as a floxed transgene or a floxed transgene expression cassette comprising a transgene encoding a therapeutic protein) for expression of a protein of interest or a fragment thereof. In preferred embodiments, the polynucleotide comprising the transgene is administered to the subject with a delivery vehicle such as a viral vector or an AAV vector (e.g., an rAAV vector).

[0368] In some embodiments, the method is intended to express a therapeutic agent (e.g., an antibody or antigen-binding fragment thereof) in a patient, thereby modulating or activating a desired biological activity.

[0369] In some embodiments, the method of treatment is intended to treat a disease or condition caused by a defect or deficiency in a single gene or single gene product (e.g., an mRNA transcript or a protein). In some embodiments, the method of treatment is intended to treat a disease or condition that leads to a defect or deficiency in multiple gene products. In some embodiments, the defect or deficiency is defined by reduced expression, reduced activity, and / or aberrant localization of the one or more gene products (e.g., an mRNA transcript or a protein).

[0370] In some embodiments, the method of treatment is intended to replace, supplement, or replenish an absent, deficient (e.g., low expression levels), or defective (e.g., mutant, loss-of-function, or low biological or catalytic activity) gene product (e.g., an mRNA transcript or a protein) in a subject having a disorder or condition. In some embodiments, the disorder or condition is characterized by a loss-of- function mutation or a gene deletion. In other embodiments, the disorder or condition is acquired (e.g., a deficient or defective gene product from stochastic or environmental factors).

[0371] In some embodiments, the transgene encodes a protein or fragment thereof that is identical (i.e., retains 100% sequence identity) to the wild-type amino acid sequence of the protein to replace, supplement, or replenish low levels a deficient or defective protein. In some embodiments, the transgene encodes a protein or fragment thereof that shares at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the wild-type amino acid sequence or a region therein to express a protein or fragment thereof that has improved biological function (e.g., increased catalytic function or reduced immunogenicity). Some examples in which a polypeptide or protein may be modified for improved biological function include mutating or adding sites for post-translational modifications (e.g., glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation, among others, or a combination thereof), mutating or adding cysteines for altered or added disulfide bonds, modifying binding sites for enhanced binding activity between the protein of interest and one or more known binding partners, modifying sites of protease binding or targeted cleavage, modifying a signal sequence for enhanced secretion or altered localization, among other protein modifications known in the art.

[0372] In some embodiments, the method of treatment is intended to increase or supplement the expression of a normally expressing protein (e.g., a protein that is present at a concentration within an accepted healthy range) in a subject having a disorder or condition that would benefit from increased expression of said protein. Increased expression of a normally expressing protein may be desired in order to increase the rate of an enzymatic reaction, enhance the potency or rate of a signaling response (e.g., PATENT

[0373] ATTORNEY DOCKET NO.: 51772-016WO2 intracellularly, or extracell ularly) , modulate trafficking or adhesion of a cell or cellular component, increase the likelihood or propensity of binding or a transient interaction to occur (e.g., based on the affinity or KD of two or more molecules), or otherwise modulate one or more biological processes. Such methods may be clinically useful for increasing the expression of a protein with a redundant function to a deficient or defective protein. Such methods may also be clinically useful for modulating a disease-causing protein or protein fragment that is logistically more difficult to employ for a method of treatment due to an assortment of non-limiting factors including large transgene size, low accessibility of a target cell or tissue, and / or high immunogenicity of the protein or fragment thereof.

[0374] In some embodiments, the method of treatment increases the expression of a protein or fragment thereof (e.g., upregulates, induces the expression of an exogenous protein or polypeptide) to modulate or regulate a separate causative agent underlying a disease. Such embodiments may be clinically useful for blocking, inhibiting, proteolyzing, mediating clearance of, or otherwise attenuating the effect of a causative agent such as a pathogen (e.g., a virus, a bacterium, a fungus, or a parasite) or a pro- inflammatory protein (e.g., a cytokine or a cytokine receptor).

[0375] In some embodiments, treatment with an effective amount of a polynucleotide comprising a transgene that encodes a protein or a pharmaceutical composition comprising the same increases the expression of the gene by about 5% to 50% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%), by about 50% to 100% (about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%), or greater than 100% (about 100%, about 125%, about 150%, about 175%, about 200%, about 225%, about 250%, about 275%, about 300%, or more) as compared to a reference such as a biological sample from a control subject or a biological sample of the same subject prior to the administration of treatment. In some embodiments, a control subject is a subject with the same disease or condition who has not received the method of treatment. In other embodiments, a control subject is a healthy control subject.

[0376] In some embodiments, treatment with an effective amount of a polynucleotide comprising a transgene that encodes a protein or a pharmaceutical composition comprising the same increases the expression of the gene by about 1 -fold, by about 2-fold, by about 3-fold, by about 4-fold, by about 5-fold, by about 6-fold, by about 7-fold, by about 8-fold, by about 9-fold, by about 10-fold, by about 11 -fold, by about 12-fold, by about 13-fold, by about 14-fold, by about 15-fold, by about 16-fold, by about 17-fold, by about 18-fold, by about 19-fold, by about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40- fold, about 45-fold, about 50-fold, or more as compared to a reference such as a biological sample from a control subject or a biological sample of the same subject prior to the administration of treatment. In some embodiments, a control subject is a subject with the same disease or condition who has not received the method of treatment. In other embodiments, a control subject is a healthy control subject.

[0377] In some embodiments, an effective amount of a polynucleotide comprising a transgene that encodes a protein or a pharmaceutical composition comprising the same corresponds to a therapeutic level of gene expression. In some embodiments, a therapeutic level is a physiologically acceptable expression level based on a healthy control subject or a median expression level based on a plurality of healthy control subjects. In some embodiments, a therapeutic level is an expression level that falls within PATENT

[0378] ATTORNEY DOCKET NO.: 51772-016WO2 a range of accepted expression levels based on subject characteristics (e.g., a subject’s age, sex, weight, height, ethnicity, lifestyle, and / or one or more comorbidities). In further embodiments, a therapeutic level is an expression level that exceeds physiological values, such that the therapeutic expression level has about a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, 500%, or more than about 500% greater relative to an expression level based on a healthy control subject or a median expression level based on a plurality of healthy control subjects.

[0379] In some embodiments, an effective amount of a polynucleotide comprising a transgene that encodes a protein or a pharmaceutical composition comprising the same corresponds to a sub- therapeutic level of gene expression. In some embodiments, a sub-therapeutic level is an expression level below a therapeutic level, such as an expression level below a physiologically acceptable expression level based on a healthy control subject or a median expression level based on a plurality of healthy control subjects. In some embodiments, a sub-therapeutic level is an expression level that falls within a range of accepted expression levels based on subject characteristics (e.g., a subject’s age, sex, weight, height, ethnicity, lifestyle, and / or one or more comorbidities). In some embodiments, a sub- therapeutic level is an expression level that is below a physiological value, such that the sub-therapeutic expression level is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% lower than a physiological value. In other embodiments, a sub-therapeutic level is an expression level that is below a therapeutic level, such that the sub-therapeutic expression level is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% lower than a therapeutic level.

[0380] In some embodiments, treatment with an effective amount of a polynucleotide comprising a transgene that encodes a protein or a pharmaceutical composition comprising the same results in ubiquitously increased expression of the gene, such there is a measurable increase across all organ systems or all tissues of the subject (e.g., throughout the body). In other embodiments, treatment with an effective amount of a polynucleotide comprising a transgene that encodes a protein or a pharmaceutical composition comprising the same results in locally increased gene expression, such that there is a measurable increase in a limited region of the body of the subject (e.g., in a subset of tissues, in one or more organs, or in one or more organ system of interest).

[0381] In some embodiments, the increased expression of the gene following the treatment is measured by an increase in mRNA transcript levels or concentrations (e.g., an mRNA transcript that corresponds to the transgene) relative to a reference. Methods of measuring mRNA transcript expression levels are routine in the art. Exemplary methods of measuring mRNA transcript expression levels include, but are not limited to, quantitative polymerase chain reaction (qPCR), reverse transcription qPCR (RT-qPCR), RNA sequencing (RNA-seq), spectrophotometric analyses, flow cytometry, in situ hybridization methods, Northern blot analyses, or an array using one or more hybridizing oligonucleotides or antibodies against one or more mRNA transcripts of interest.

[0382] In some embodiments, the increased expression of the gene following the treatment is measured by an increase in protein expression levels or concentrations (e.g., a protein encoded by the transgene) relative to a reference. Methods of measuring protein expression levels are routine in the art. Exemplary PATENT

[0383] ATTORNEY DOCKET NO.: 51772-016WO2 methods of measuring protein expression levels include, but are not limited to, Western blot analysis, an enzyme-linked immunosorbent assay (ELISA), mass spectrometry, flow cytometry, fluorimetry, colorimetry, spectrophotometric analyses, or an array using a binding oligonucleotide (e.g., an aptamer) or an antibody against one or more protein of interest.

[0384] In some embodiments, the increased expression of the transgene that encodes a protein is sustained following administration. In some embodiments, in the absence of the gene expression silencer, the increased expression is sustained indefinitely following administration. In some embodiments, the increased expression is sustained for at least one week following administration. In some embodiments, the increased expression is sustained between one week and 10 years following administration. In some embodiments, the increased expression is sustained between 1 week and 7 years following administration. In some embodiments, the increased expression is sustained between 1 week and 5 years following administration. In some embodiments, the increased expression is sustained between 1 week and 3 years following administration. In some embodiments, the increased expression is sustained between 1 week and 1 year following administration.

[0385] In some embodiments, the increased expression is sustained between 1 week and 24 months following administration. In some embodiments, the increased expression is sustained between 1 week and 23 months following administration. In some embodiments, the increased expression is sustained between 1 week and 22 months following administration. In some embodiments, the increased expression is sustained between 1 week and 21 months following administration. In some embodiments, the increased expression is sustained between 1 week and 20 months following administration. In some embodiments, the increased expression is sustained between 1 week and 19 months following administration. In some embodiments, the increased expression is sustained between 1 week and 18 months following administration. In some embodiments, the increased expression is sustained between 1 week and 17 months following administration. In some embodiments, the increased expression is sustained between 1 week and 16 months following administration. In some embodiments, the increased expression is sustained between 1 week and 15 months following administration. In some embodiments, the increased expression is sustained between 1 week and 14 months following administration. In some embodiments, the increased expression is sustained between 1 week and 13 months following administration. In some embodiments, the increased expression is sustained between 1 week and 12 months following administration. In some embodiments, the increased expression is sustained between 1 week and 11 months following administration. In some embodiments, the increased expression is sustained between 1 week and 10 months following administration. In some embodiments, the increased expression is sustained between 1 week and 9 months following administration. In some embodiments, the increased expression is sustained between 1 week and 8 months following administration. In some embodiments, the increased expression is sustained between 1 week and 7 months following administration. In some embodiments, the increased expression is sustained between 1 week and 6 months following administration. In some embodiments, the increased expression is sustained between 1 week and 5 months following administration. In some embodiments, the increased expression is sustained between 1 week and 4 months following administration. In some embodiments, the increased expression is sustained between 1 week and 3 months following administration. In some embodiments, PATENT

[0386] ATTORNEY DOCKET NO.: 51772-016WO2 the increased expression is sustained between 1 week and 2 months following administration. In some embodiments, the increased expression is sustained between 1 week and 1 month following administration.

[0387] In some embodiments, the increased expression is sustained for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks, at least 16 weeks, at least 17 weeks, at least 18 weeks, at least 19 weeks, at least 20 weeks, at least 21 weeks, at least 22 weeks, at least 23 weeks, at least 24 weeks, at least 25 weeks, or at least 26 weeks following administration.

[0388] In some embodiments, the increased expression is sustained for more than 10 years following administration. In some embodiments, the increased expression is sustained between 6 months and 10 years following administration. In some embodiments, the increased expression is sustained between 1 year and 10 years following administration. In some embodiments, the increased expression is sustained between 2 years and 10 years following administration. In some embodiments, the increased expression is sustained between 3 years and 10 years following administration. In some embodiments, the increased expression is sustained between 4 years and 10 years following administration. In some embodiments, the increased expression is sustained between 4 years and 10 years following administration. In some embodiments, the increased expression is sustained between 5 years and 10 years following administration. In some embodiments, the increased expression is sustained between 6 years and 10 years following administration. In some embodiments, the increased expression is sustained between 7 years and 10 years following administration. In some embodiments, the increased expression is sustained between 8 years and 10 years following administration. In some embodiments, the increased expression is sustained between 9 years and 10 years following administration.

[0389] In some embodiments, expression of the transgene that encodes the protein of interest is sustained within fifty percent of an initial expression level. In some embodiments, expression of the transgene that encodes the protein of interest is sustained within forty-five percent of an initial expression level. In some embodiments, expression of the transgene that encodes the protein of interest is sustained within forty percent of an initial expression level. In some embodiments, expression of the transgene that encodes the protein of interest is sustained within thirty-five percent of an initial expression level. In some embodiments, expression of the transgene that encodes the protein of interest is sustained within thirty percent of an initial expression level. In some embodiments, expression of the transgene that encodes the protein of interest is sustained within twenty-five percent of an initial expression level. In some embodiments, expression of the transgene that encodes the protein of interest is sustained within twenty percent of an initial expression level. In some embodiments, expression of the transgene that encodes a protein of interest is sustained within fifteen percent of an initial expression level. In some embodiments, expression of the transgene that encodes a protein of interest is sustained within ten percent of an initial expression level. In some embodiments, expression of the transgene that encodes a protein of interest is sustained within nine percent, eight percent, seven percent, six percent, five percent, four percent, three percent, two percent, or one percent of an initial expression level.

[0390] The magnitude or duration of the increased expression of the mRNA transcript and / or protein PATENT

[0391] ATTORNEY DOCKET NO.: 51772-016WO2 encoded by the transgene following administration of the polynucleotide or pharmaceutical composition comprising the same may depend on one or more factors. Such factors may include, but are not limited to, the route of administration, the dose, stability of the mRNA transcript and / or protein, or the disease or condition for which the treatment is intended, and / or one or more comorbidities of the subject.

[0392] As described in the foregoing section, in some embodiments, the expression of the mRNA transcript and / or protein encoded by the transgene is terminated by administration of Cre recombinase (e.g., LNP-Cre, a Cre recombinase administered using any other Cre delivery technique described herein, or a nucleic acid encoding Cre recombinase administered using any other Cre delivery technique described herein).

[0393] In some embodiments, the method of treatment includes administering an effective amount of a polynucleotide comprising a transgene or a pharmaceutical composition comprising the same to the subject (e.g., a human) via in vivo, in vitro, or ex vivo methods of administration, or any combination thereof. In some embodiments, the method of treatment includes in vivo methods of administration, such that the polynucleotide or the pharmaceutical composition is administered directly to the body of the subject by an appropriate route of administration, such as one or more of the methods described below. In some embodiments, the method of treatment includes in vitro or ex vivo methods of administration, such that the polynucleotide or pharmaceutical composition containing the same is delivered to one or more isolated cells or tissues from the subject and then optionally separately implanted, dispensed, or deposited to the body of the subject.

[0394] The method of treatment includes administering an effective amount of a polynucleotide comprising a transgene for expression of a protein of interest or a fragment thereof or a pharmaceutical composition comprising the same to the subject (e.g., a human) by any appropriate route of administration. Appropriate routes of administration include, but are not limited to, intramuscular (IM), subcutaneous, intradermal, intravenous, intraperitoneal, intraarterial, intraventricular, intranasal, intraorbital, intracranial, or intraosseous administration. In some embodiments, the method of treatment includes administering one codon optimized gene product or a pharmaceutical composition comprising the same to a subject to produce a therapeutic effect.

[0395] In some embodiments, an effective amount of a polypeptide comprising a transgene or a pharmaceutical composition comprising the same is administered to a subject between 1 and 10 times. In some embodiments, an effective amount of a polypeptide comprising a transgene or a pharmaceutical composition comprising the same is administered to a subject between 1 and 5 times. In further embodiments, an effective amount of a polypeptide comprising a transgene or a pharmaceutical composition comprising the same is administered to a subject between 3 and 5 times. In further embodiments, an effective amount of a polypeptide comprising a transgene or a pharmaceutical composition comprising the same is administered to a subject between 1 and 3 times.

[0396] B. Using Recombinase Intervention to Reduce Expression of a Previously Administered Gene of Interest PATENT

[0397] ATTORNEY DOCKET NO.: 51772-016WO2

[0398] (i) Terminating transgene expression in patients that become contraindicated for a previously administered gene of interest

[0399] Following the administration of an effective amount of one or more polynucleotides comprising a transgene (such as a floxed transgene or a floxed transgene expression cassette, in each case comprising a transgene encoding a therapeutic protein) or one or more pharmaceutical compositions comprising the same, it may be desirable to terminate expression of the transgene because the patient becomes contraindicated for the transgene that has been previously delivered. In this regard, “termination” of transgene expression refers to a reduction in transgene expression to a level that is beneath that which is necessary for the transgene to exhibit a biological effect in the patient. For example, expression of the transgene may be terminated, as this term is used herein, by reducing expression of the transgene to a level that is insufficient to exhibit an associated phenotype - either systemically or in a specific cell type, tissue, or organ of interest - even though some residual expression of the transgene may be detected using a conventional protein or nucleic acid detection assay, particularly if the assay has a low limit of detection. For the avoidance of doubt, it is to be understood that in some embodiments, transgene expression may be terminated by eliminating expression of the transgene altogether, such that expression of the transgene is not detectable at the level of the protein and / or corresponding nucleic acid. However, this need not be the case in order for effective transgene suppression to occur using the composition and methods of the disclosure, as transgene expression may be deemed to be sufficiently terminated once the expression level falls to a level beneath which one or more biological effects associated with the encoded gene product no longer manifest. Examples of such biological effects include the molecular activity associated with the corresponding gene product (e.g., in the case of an enzyme, the corresponding conversion of the substrate to its biochemical product), as well as an effect that the gene product may induce upon expression in the patient’s body (e.g., an immune response mounted by the patient’s immune system on the gene product, as may be the case for a transgene that the patient does not express endogenously and for which the patient’s immune system has not been tolerized.

[0400] Situations in which it may be beneficial to terminate transgene expression in a subject following administration of a gene therapy agent include those in which the subject has or develops a contraindication (e.g., chronic or long-lasting contraindication) for the previously provided transgene. As a non-limiting example, silencing the transgene after a certain period of time using the Cre-lox recombination systems described herein can be particularly advantageous in patients for whom the administration of a gene therapy agent (e.g., a therapeutic transgene encoding a therapeutic protein) subsequently resulted in an immunogenic response (e.g., a chronic immunogenic response) against the therapeutic product, such as an immunogenic response against the encoded protein once the protein was expressed. This can occur, for instance, if the patient has never had prior exposure to the encoded protein. In circumstances such as these, the patient’s immune system has not been tolerized against the encoded protein. Accordingly, there exists the risk that, upon expression of the encoded protein, the patient’s immune cells (e.g., cross-reactive T cells and / or B cells) may mount an immune response against the encoded protein because the protein is recognized as foreign. In instances such as these, it may be beneficial to terminate expression of the previously administered transgene such that expression PATENT

[0401] ATTORNEY DOCKET NO.: 51772-016WO2 of the encoded protein falls beneath a level that is necessary for the immune response to cease to a degree that no longer adversely affects the patient. As is explained above, this may be by complete termination of transgene expression to a level of protein that is substantially undetectable or, alternatively, to a level of residual expression that is not significant enough to induce a harmful immune response.

[0402] Using the Cre-lox recombination system described herein to terminate transgene expression can also be beneficial, for example, in patients for whom the therapeutic product (e.g., a therapeutic protein) accumulates to levels that have induced toxicity in the patient, either by way of on-target or off-target effects. As a result of the long duration of expression of the gene therapy modalities described herein, there may be instances in which the encoded product of the transgene of interest reaches levels that are unsafe, not tolerated, or otherwise detrimental to the patient’s health. In examples such as this, it may be advantageous to terminate gene expression (or, alternatively, to reduce gene expression to a safe or tolerable level, as is explained in section IV(B)(ii), below).

[0403] Additional examples of circumstances in which it may be beneficial to terminate expression of a previously provided transgene include instances in which, following administration of a gene therapy agent, a patient commences with a treatment regimen (e.g., a chronic treatment regimen) that, in turn, is contraindicated for the previously administered transgene of interest. For instance, following administration of a gene therapy product, a patient may develop a chronic infection, necessitating treatment with an antiviral, antibacterial, or other antimicrobial agent that is contraindicated for the previously administered transgene. Similarly, in another example, following administration of a gene therapy product, a patient may be diagnosed with or develop a cancer or other cell proliferation disorder, necessitating treatment with, e.g., a chemotherapy and / or immunotherapy regimen. If these regimens are contraindicated with the originally administered transgene, one can desirably terminate expression of the previously administered transgene using the Cre-lox paradigm disclosed herein.

[0404] Yet another example of a circumstance that may warrant termination of a previously administered transgene in an instance in which patient develops an autoimmune disease or undergoes an organ transplant procedure following administration of a transgene of interest. As a result of the patient’s new condition, the patient may necessitate treatment (e.g., chronic treatment) with one or more immunosuppressive agents. This new treatment regimen, triggered by the patient’s new circumstances, may be contraindicated for the originally administered transgene of interest (which may, as an example, have encoded a protein product that is intended to stimulate the patient’s immune system). In these instances, the patient may benefit from treatment with the Cre-lox-mediated transgene attenuation methodology described herein.

[0405] In still a further example, a patient may be diagnosed with or develop a metabolic disorder following administration of the originally delivered transgene of interest. For example, following administration of the previously provided gene therapy agent, the patient may be diagnosed with or develop obesity, hypercholesterolemia, hypertension, among others, all of which are conditions that, in turn, may necessitate treatment with one or more therapeutic interventions. If these intervention regimens are contraindicated for the original transgene of interest, the patient may desirably be treated with the Cre-lox system described herein in order to terminate expression of the transgene. PATENT

[0406] ATTORNEY DOCKET NO.: 51772-016WO2

[0407] For any instance in which a patient undergoes treatment with a Cre-lox system of the disclosure in order to effectively terminate expression of the previously provided transgene, the patient may be administered the recombinase enzyme in one or more doses that, together, provide a level of the recombinase that is sufficient to terminate expression of the target transgene, as the term “terminate” is used herein. For example, the recombinase may be administered in a single dose that, alone, is sufficient to reduce expression of the transgene to such a level as to overcome the contraindication and mitigate the risk posed by the interaction between the original transgene and the patient’s new condition, lifestyle, and / or therapeutic intervention. Alternatively, the patient may be administered a plurality of doses that, together, are sufficient to terminate expression of the transgene (i.e., a plurality of doses that, when combined, are sufficient to reduce expression of the transgene to such a level as to overcome the contraindication and mitigate the risk posed by the interaction between the original transgene and the patient’s new condition, lifestyle, and / or therapeutic intervention). In some embodiments, each dose is separated by at least one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, or ten weeks.

[0408] The ability to iteratively dose a patient with a recombinase enzyme in order to terminate expression of the target transgene arises out of a significant advantage exhibited by recombinase delivery systems of the disclosure, particularly those involving LNPs: unlike certain viral particles, which may have a tendency to induce immunogenic reactions to viral capsid or envelope proteins upon administration to a patient, LNPs are substantially less likely to trigger an immune response. Accordingly, LNP systems for delivering a recombinase of the disclosure can be administered to a patient in a series of repeat doses without incurring the same risk of an immunogenic response as would be incurred by certain viral delivery modalities.

[0409] This beneficial property of LNPs allows patients that are in need of transgene termination to be administered multiple, sequential doses in order to reach the level of recombinase expression that is sufficient to suppress transgene expression to a level that no longer poses a threat to the patient’s health. For instance, a small, initial dose of the recombinase may be administered to the patient at a starting timepoint, and following a waiting period, the patient may be evaluated to determine whether expression of the transgene has been sufficiently terminated. If it has, the patient may not require further administration of the recombinase. Alternatively, if expression of the transgene has not sufficiently terminated, the patient may be administered a subsequent dosage of the recombinase. Following this subsequent dosage, the patient may be similarly evaluated to determine whether expression of the transgene has now been sufficiently terminated so as to mitigate the health risk at hand. This process of sequential dosing and evaluation may be repeated, as needed, until expression of the transgene has reached a level that obviates the contraindication brought on by the patient’s new condition.

[0410] The recombinase (e.g., Cre recombinase) may be administered to the patient at any timepoint following administration of the original gene therapy product, as needed based on the patient’s new condition, lifestyle, and / or therapeutic intervention regimen.

[0411] For example, in some embodiments, the recombinase (e.g., Cre recombinase) is administered at least one week following administration of the originally provided transgene. In some embodiments, the recombinase is administered between one week and 10 years following administration of the originally PATENT

[0412] ATTORNEY DOCKET NO.: 51772-016WO2 provided transgene. In some embodiments, the recombinase is administered between 1 week and 7 years following administration of the originally provided transgene. In some embodiments, the recombinase is administered between 1 week and 5 years following administration of the originally provided transgene. In some embodiments, the recombinase is administered between 1 week and 3 years following administration of the originally provided transgene. In some embodiments, the recombinase is administered between 1 week and 1 year following administration of the originally provided transgene.

[0413] In some embodiments, the recombinase is administered between 1 week and 24 months following administration of the originally provided transgene. In some embodiments, the recombinase is administered between 1 week and 23 months following administration of the originally provided transgene. In some embodiments, the recombinase is administered between 1 week and 22 months following administration of the originally provided transgene. In some embodiments, the recombinase is administered between 1 week and 21 months following administration of the originally provided transgene. In some embodiments, the recombinase is administered between 1 week and 20 months following administration of the originally provided transgene. In some embodiments, the recombinase is administered between 1 week and 19 months following administration of the originally provided transgene. In some embodiments, the recombinase is administered between 1 week and 18 months following administration of the originally provided transgene. In some embodiments, the recombinase is administered between 1 week and 17 months following administration of the originally provided transgene. In some embodiments, the recombinase is administered between 1 week and 16 months following administration of the originally provided transgene. In some embodiments, the recombinase is administered between 1 week and 15 months following administration of the originally provided transgene. In some embodiments, the recombinase is administered between 1 week and 14 months following administration of the originally provided transgene. In some embodiments, the recombinase is administered between 1 week and 13 months following administration of the originally provided transgene. In some embodiments, the recombinase is administered between 1 week and 12 months following administration of the originally provided transgene. In some embodiments, the recombinase is administered between 1 week and 11 months following administration of the originally provided transgene. In some embodiments, the recombinase is administered between 1 week and 10 months following administration of the originally provided transgene. In some embodiments, the recombinase is administered between 1 week and 9 months following administration of the originally provided transgene. In some embodiments, the recombinase is administered between 1 week and 8 months following administration of the originally provided transgene. In some embodiments, the recombinase is administered between 1 week and 7 months following administration of the originally provided transgene. In some embodiments, the recombinase is administered between 1 week and 6 months following administration of the originally provided transgene. In some embodiments, the recombinase is administered between 1 week and 5 months following administration of the originally provided transgene. In some embodiments, the recombinase is administered between 1 week and 4 months following administration of the originally provided transgene. In some embodiments, the recombinase is administered between 1 week and 3 months following administration of the originally provided transgene. PATENT

[0414] ATTORNEY DOCKET NO.: 51772-016WO2

[0415] In some embodiments, the recombinase is administered between 1 week and 2 months following administration of the originally provided transgene. In some embodiments, the recombinase is administered between 1 week and 1 month following administration of the originally provided transgene.

[0416] In some embodiments, the Cre-lox systems of the disclosure may be used to terminate (as that term is used herein) transgene expression nonspecifically throughout the patient’s body. Alternatively, the Cre-lox systems of the disclosure may be used to terminate (as that term is used herein) transgene expression in one or more specific cell types, tissues, tissue systems, organs, or organ systems in the patient.

[0417] For example, in some embodiments, the recombinase (e.g., as a protein per se and / or as a nucleic acid encoding the same, such as a DNA or mRNA polynucleotide encoding the same) may be administered directly to one or more cells, tissues, or organs at the exclusion of others, thereby restricting expression of the recombinase (and subsequent reduction of transgene expression) to specific cell types, tissues, tissue systems, organs, or organ systems. Additionally or alternatively, the recombinase may be administered as a nucleic acid (e.g., a DNA polynucleotide, such as a viral vector containing the same) encoding the recombinase enzyme such that the open reading frame encoding the recombinase is operably linked to a gene regulation element that is specifically active in a desired cell type, tissue, tissue system, organ, or organ system. And in still further examples, the recombinase may be administered in the form of a viral vector having a tropism that is specific for one or more cell types, tissues, tissue systems, organs, or organ systems, thereby directing expression of the recombinase (and consequent termination of transgene expression) to a desired region of the patient’s body.

[0418] In some embodiments, such as those instances in which the expression of the transgene is reduced to a level that is beneath that required to elicit an adverse event in the patient, reduction in transgene expression may be evaluated using any nucleic acid or protein detection method known in the art. For example, the decrease in transgene expression may be evaluated by detecting a decrease in mRNA transcript levels or concentrations. Methods of measuring mRNA transcript expression levels include conventional mRNA detection and quantification assays known in the field, including, e.g., quantitative polymerase chain reaction (qPCR), reverse transcription qPCR (RT-qPCR), RNA sequencing (RNA-seq), spectrophotometric analyses, flow cytometry, in situ hybridization methods, Northern blot analyses, or an array using one or more hybridizing oligonucleotides or antibodies against one or more mRNA transcripts of interest. In any of these settings, the reduction in transgene expression may simply be a reduction to beneath a threshold that is required for triggering a harmful reaction in the patient, or, alternatively, the reduction in transgene expression may be to a level that is substantially undetectable using the assay of choice.

[0419] In some embodiments, the reduction in transgene expression may be measured by evaluating a reduction in the level of protein encoded by the target transgene. Exemplary methods of measuring protein expression levels include, but are not limited to, Western blot analysis, an enzyme-linked immunosorbent assay (ELISA), mass spectrometry, flow cytometry, fluorimetry, colorimetry, spectrophotometric analyses, or an array using a binding oligonucleotide (e.g., an aptamer) or an antibody against one or more proteins of interest. Here again, the reduction in transgene expression may simply be a reduction to beneath a threshold that is required for triggering a harmful reaction in the PATENT

[0420] ATTORNEY DOCKET NO.: 51772-016WO2 patient, or, alternatively, the reduction in transgene expression may be to a level that is substantially undetectable using the assay of choice.

[0421] In some embodiments, administration of the recombinase reduces the expression of the protein of interest (e.g., the protein encoded by the target transgene) by about 2-fold, by about 3-fold, by about 4- fold, by about 5-fold, by about 6-fold, by about 7-fold, by about 8-fold, by about 9-fold, by about 10-fold, by about 15-fold, by about 20-fold, by about 25-fold, by about 30-fold, by about 35-fold, by about 40-fold, by about 45-fold, by about 50-fold, or greater than 50-fold, as compared to expression of the protein of interest in the subject prior to administration of the recombinase. In some embodiments, administration of the recombinase reduces the expression of the protein of interest by about 2-fold, by about 3-fold, by about 4-fold, by about 5-fold, by about 6-fold, by about 7-fold, by about 8-fold, by about 9-fold, by about 10-fold, by about 11 -fold, by about 12-fold, by about 13-fold, by about 14-fold, by about 15-fold, by about

[0422] 16-fold, by about 17-fold, by about 18-fold, by about 19-fold, by about 20-fold, by about 21 -fold, by about

[0423] 22-fold, by about 23-fold, by about 24-fold, by about 25-fold, by about 26-fold, by about 27-fold, by about

[0424] 28-fold, by about 29-fold, by about 30-fold, by about 31 -fold, by about 32-fold, by about 33-fold, by about

[0425] 34-fold, by about 35-fold, by about 36-fold, by about 37-fold, by about 38-fold, by about 39-fold, by about

[0426] 40-fold, by about 41 -fold, by about 42-fold, by about 43-fold, by about 44-fold, by about 45-fold, by about

[0427] 46-fold, by about 47-fold, by about 48-fold, by about 49-fold, by about 50-fold, or by greater than 50-fold, as compared to expression of the protein of interest in the subject prior to administration of the recombinase.

[0428] In some embodiments, the terminated expression of the transgene that encodes a protein is sustained for a given amount of time following administration. In some embodiments, the terminated expression is sustained for the remainder of the subject’s life following administration.

[0429] In some embodiments, the method of treatment includes delivering an effective amount of a polynucleotide comprising a floxed transgene or a floxed transgene expression cassette comprising a transgene encoding a therapeutic protein (for expression of a protein of interest or a fragment thereof) or a pharmaceutical composition comprising the same via in vivo, in vitro, or ex vivo methods of delivery, or any combination thereof. In some embodiments, the method of treatment includes in vivo methods of administration, such that the polynucleotide or the pharmaceutical composition is administered directly to the body of the subject. In some embodiments, the method of treatment includes in vitro or ex vivo methods of administration, such that the polynucleotide or pharmaceutical composition comprising the same is delivered to one or more isolated cells or tissues from the subject and then separately implanted, dispensed, or deposited to the body of the subject.

[0430] The method of treatment may include administering an effective amount of a recombinase (e.g., Cre recombinase) or a pharmaceutical composition comprising the same to the subject (e.g., a human) by any appropriate route of administration. Appropriate routes of administration include, but are not limited to, intramuscular (IM), subcutaneous, intradermal, intravenous, intraperitoneal, intraarterial, intraventricular, intranasal, intraorbital, intracranial, or intraosseous administration. In some embodiments, the method of treatment includes administering one codon optimized gene product or a pharmaceutical composition comprising the same to a subject to produce a therapeutic effect.

[0431] In exemplary embodiments, the route of administration of a polynucleotide comprising the floxed PATENT

[0432] ATTORNEY DOCKET NO.: 51772-016WO2 transgene (or a floxed transgene expression cassette comprising a transgene encoding a therapeutic protein) and a Cre recombinase or pharmaceutical compositions thereof are the same. In further embodiments, a polynucleotide comprising a floxed transgene (or a floxed transgene expression cassette comprising a transgene encoding a therapeutic protein) and a Cre recombinase or pharmaceutical compositions thereof are administered within a certain distance of a region of the body of a subject in need thereof (e.g., a human). In some embodiments, a polynucleotide comprising a floxed transgene (or a floxed transgene expression cassette comprising a transgene encoding a therapeutic protein) and a Cre recombinase or pharmaceutical compositions thereof are administered within a 10 cm distance or closer (e.g., within a 10 cm distance, within a 9 cm distance, within an 8 cm distance, within a 7 cm distance, within a 6 cm distance, within a 5 cm distance, within a 4 cm distance, within a 3 cm distance, within a 2 cm distance, within a 1 cm distance). In some embodiments, a polynucleotide comprising a floxed transgene (or a floxed transgene expression cassette comprising a transgene encoding a therapeutic protein) and a Cre recombinase or pharmaceutical compositions thereof are administered within a 3 cm distance or closer (e.g., within a 3 cm distance, within a 2 cm distance, within a 1 cm distance). In further embodiments, a polynucleotide comprising a floxed transgene (or a floxed transgene expression cassette comprising a transgene encoding a therapeutic protein) and a Cre recombinase or pharmaceutical compositions thereof are administered within 1 cm (e.g., within 1 cm, within 0.9 cm, within 0.8 cm, within 0.7 cm, within 0.6 cm, within 0.5 cm, within 0.4 cm, within 0.3 cm, or closer). In some embodiments, the location of the body of the subject for administration of a polynucleotide comprising a floxed transgene (or a floxed transgene expression cassette comprising a transgene encoding a therapeutic protein) and a Cre recombinase or pharmaceutical compositions comprising the same are marked prior to administration, e.g., by a medical tattoo.

[0433] (ii) Modulating transgene expression in patients if transgene expression levels exceed a desired therapeutic window

[0434] In addition to being used to terminate transgene expression, as is outlined in the section above, the compositions and methods of the disclosure can also be used to reduce transgene expression to within a desired expression level range if, for example, following the administration of a transgene of interest, the expression level exceeds that which is necessary to exhibit the desired therapeutic phenotype and / or exceeds the expression level that is safely tolerated by the patient without inducing toxic side effects (e.g., adverse events due to on-target or off-target interactions).

[0435] For instance, the compositions and methods of the disclosure may be used to reduce the expression level of a previously provided transgene of interest into a therapeutic window if the transgene was originally provided to the patient in an amount such that the resulting expression level of the encoded product is higher than that which is needed for the intended therapeutic effect to manifest. In situations such as this, the recombinase systems described herein may be provided to the patient in a “subsaturating” dose so as to not terminate expression of the transgene, as that term is used in the section above, but instead to down-titrate the expression level of the encoded transgene product so as to fit within a desired range that is necessary and / or useful for achieving a therapeutic effect. PATENT

[0436] ATTORNEY DOCKET NO.: 51772-016WO2

[0437] In another example, the compositions and methods of the disclosure may be used to reduce the expression level of a previously provided transgene of interest into a therapeutic window if the transgene was originally provided to the patient in an amount such that the resulting expression level of the encoded product is higher than that which is safely tolerated by the patient without effectuating adverse events. Here again, in such situations, the recombinase systems described herein may be provided to the patient in a sub-saturating dose so as to not terminate expression of the transgene, but instead to modulate the expression level of the encoded transgene product so as to fit within a range that engenders the intended therapeutic effect without incurring toxicity.

[0438] For any instance in which a patient undergoes treatment with a Cre-lox system of the disclosure in order to down-titrate expression of the previously provided transgene, the patient may be administered the recombinase enzyme in one or more doses that, together, provide a sub-saturating level of the recombinase that is sufficient to reduce expression of the target transgene. For example, the recombinase may be administered in a single, sub-saturating dose that, alone, is sufficient to down-titrate expression of the transgene to such a level that no longer poses a risk to the patient. Alternatively, the patient may be administered a plurality of doses that, together, are sufficiently sub-saturating as to reduce expression of the transgene (i.e. , a plurality of doses that, when combined, are sufficient to down-titrate expression of the transgene to such a level as to no longer pose the risk to the patient that is incurred by higher transgene expression levels).

[0439] Just as is described in the section above, the ability to iteratively dose a patient with a recombinase enzyme in order to down-titrate expression of the target transgene arises out of the advantageous lack of immunogenicity exhibited by particular recombinase delivery systems, such as LNPs. In light of this substantial benefit, LNP systems for delivering a recombinase of the disclosure can be administered to a patient in a series of repeat doses without incurring the same risk of an immunogenic response as that brought on by viral delivery vehicles.

[0440] Accordingly, LNP-Cre delivery systems of the disclosure may be administered either as a single, sub-saturating dose or in multiple, sequential doses in order to reach the level of recombinase expression that is sufficient to down-titrate transgene expression to within a desired therapeutic window. For instance, a small, initial dose of the recombinase may be administered to the patient at a starting timepoint, and following a waiting period, the patient may be evaluated to determine whether expression of the transgene has been sufficiently reduced so as to be within a range that is simultaneously safe and therapeutically effective. If it has, the patient may not require further administration of the recombinase. Alternatively, if expression of the transgene has not been sufficiently down-titrated, the patient may be administered a subsequent dosage of the recombinase. Following this subsequent dosage, the patient may be similarly evaluated to determine whether expression of the transgene has now been sufficiently reduced so as to mitigate the adverse event risk at hand. This process of sequential dosing and evaluation may be repeated, as needed, until expression of the transgene has reached a level that is deemed safe and effective for the underlying disease or condition being treated.

[0441] In some embodiments, the Cre-lox systems of the disclosure may be used to down-titrate transgene expression nonspecifically throughout the patient’s body. Alternatively, the Cre-lox systems of PATENT

[0442] ATTORNEY DOCKET NO.: 51772-016WO2 the disclosure may be used to down-titrate transgene expression in one or more specific cell types, tissues, tissue systems, organs, or organ systems in the patient.

[0443] For example, in some embodiments, the recombinase (e.g., as a protein per se and / or as a nucleic acid encoding the same, such as a DNA or mRNA polynucleotide encoding the same) may be administered directly to one or more cells, tissues, or organs at the exclusion of others, thereby restricting expression of the recombinase (and subsequent down-titration of transgene expression) to specific cell types, tissues, tissue systems, organs, or organ systems. Additionally or alternatively, the recombinase may be administered as a nucleic acid (e.g., a DNA polynucleotide, such as a viral vector containing the same) encoding the recombinase enzyme such that the open reading frame encoding the recombinase is operably linked to a gene regulation element that is specifically active in a desired cell type, tissue, tissue system, organ, or organ system. And in still further examples, the recombinase may be administered in the form of a viral vector having a tropism that is specific for one or more cell types, tissues, tissue systems, organs, or organ systems, thereby directing expression of the recombinase (and consequent down-titration of transgene expression) to a desired region of the patient’s body.

[0444] In some embodiments, the appropriate level of transgene expression is confirmed using a nucleic acid detection assay, such as an mRNA detection assay known in the art or described herein. Exemplary methods of measuring mRNA transcript expression levels include, without limitation, quantitative polymerase chain reaction (qPCR), reverse transcription qPCR (RT-qPCR), RNA sequencing (RNA-seq), spectrophotometric analyses, flow cytometry, in situ hybridization methods, Northern blot analyses, or an array using one or more hybridizing oligonucleotides or antibodies against one or more mRNA transcripts of interest.

[0445] In some embodiments, the appropriate level of transgene expression is confirmed using a protein detection assay, such as a protein detection assay known in the art or described herein. Exemplary methods of measuring protein expression levels include, but are not limited to, Western blot analysis, an enzyme-linked immunosorbent assay (ELISA), mass spectrometry, flow cytometry, fluorimetry, colorimetry, spectrophotometric analyses, or an array using a binding oligonucleotide (e.g., an aptamer) or an antibody against one or more proteins of interest.

[0446] In some embodiments, the method of treatment includes delivering an effective amount of a polynucleotide comprising a floxed transgene or a floxed transgene expression cassette comprising a transgene encoding a therapeutic protein (for expression of a protein of interest or a fragment thereof) or a pharmaceutical composition comprising the same via in vivo, in vitro, or ex vivo methods of delivery, or any combination thereof. In some embodiments, the method of treatment includes in vivo methods of administration, such that the polynucleotide or the pharmaceutical composition is administered directly to the body of the subject. In some embodiments, the method of treatment includes in vitro or ex vivo methods of administration, such that the polynucleotide or pharmaceutical composition comprising the same is delivered to one or more isolated cells or tissues from the subject and then separately implanted, dispensed, or deposited to the body of the subject.

[0447] The method of treatment may include administering an effective amount of a recombinase (e.g., Cre recombinase) or a pharmaceutical composition comprising the same to the subject (e.g., a human) by any appropriate route of administration. Appropriate routes of administration include, but are not limited PATENT

[0448] ATTORNEY DOCKET NO.: 51772-016WO2 to, intramuscular (IM), subcutaneous, intradermal, intravenous, intraperitoneal, intraarterial, intraventricular, intranasal, intraorbital, intracranial, or intraosseous administration. In some embodiments, the method of treatment includes administering one codon optimized gene product or a pharmaceutical composition comprising the same to a subject to produce a therapeutic effect.

[0449] In exemplary embodiments, the route of administration of a polynucleotide comprising the floxed transgene (or a floxed transgene expression cassette comprising a transgene encoding a therapeutic protein) and a Cre recombinase or pharmaceutical compositions thereof are the same. In further embodiments, a polynucleotide comprising a floxed transgene (or a floxed transgene expression cassette comprising a transgene encoding a therapeutic protein) and a Cre recombinase or pharmaceutical compositions thereof are administered within a certain distance of a region of the body of a subject in need thereof (e.g., a human). In some embodiments, a polynucleotide comprising a floxed transgene (or a floxed transgene expression cassette comprising a transgene encoding a therapeutic protein) and a Cre recombinase or pharmaceutical compositions thereof are administered within a 10 cm distance or closer (e.g., within a 10 cm distance, within a 9 cm distance, within an 8 cm distance, within a 7 cm distance, within a 6 cm distance, within a 5 cm distance, within a 4 cm distance, within a 3 cm distance, within a 2 cm distance, within a 1 cm distance). In some embodiments, a polynucleotide comprising a floxed transgene (or a floxed transgene expression cassette comprising a transgene encoding a therapeutic protein) and a Cre recombinase or pharmaceutical compositions thereof are administered within a 3 cm distance or closer (e.g., within a 3 cm distance, within a 2 cm distance, within a 1 cm distance). In further embodiments, a polynucleotide comprising a floxed transgene (or a floxed transgene expression cassette comprising a transgene encoding a therapeutic protein) and a Cre recombinase or pharmaceutical compositions thereof are administered within 1 cm (e.g., within 1 cm, within 0.9 cm, within 0.8 cm, within 0.7 cm, within 0.6 cm, within 0.5 cm, within 0.4 cm, within 0.3 cm, or closer). In some embodiments, the location of the body of the subject for administration of a polynucleotide comprising a floxed transgene (or a floxed transgene expression cassette comprising a transgene encoding a therapeutic protein) and a Cre recombinase or pharmaceutical compositions comprising the same are marked prior to administration, e.g., by a medical tattoo.

[0450] V. Kits

[0451] The compositions and methods described herein can be provided in a kit for use in expressing a transgene and / or subsequently terminating the expression. In some embodiments, the compositions and methods described herein can be provided in a kit for use in treating a disease or condition. In some embodiments, the kit may include a package insert that instructs a user of the kit to construct or synthesize a polynucleotide comprising a transgene, a vector comprising the same, a Cre recombinase (such as LNP-Cre, or a Cre recombinase or a nucleic acid encoding Cre recombinase administered using any other Cre delivery technique described herein), or a pharmaceutical composition described herein. In further embodiments, the kit may include a package insert that instructs a user of the kit to perform any one of the methods of treatment described herein. The kit may optionally include a syringe or device for administering the compositions of the present disclosure. In some embodiments, the kit may include one or more additional therapeutic agents. In some embodiments, the kit includes one or more antibodies or binding molecules to detect the expression or activity of an mRNA transcript or a protein of interest. PATENT

[0452] ATTORNEY DOCKET NO.: 51772-016WO2

[0453] EXAMPLES

[0454] Example 1 : Sustained expression of a transgene delivered with an AAV vector by intramuscular administration

[0455] Objective

[0456] The objective of this study was to characterize expression of a transgene delivered with an AAV vector by intramuscular administration.

[0457] Materials and Methods

[0458] An AAV vector construct was produced, in which the genome contained a GLP-1 transgene. The construct also included a cmv promoter and a polyadenylation signal sequence (pA). The AAV capsid for the vector was AAV9.

[0459] Results

[0460] The AAV vector construct containing GLP-1 was delivered to Rag mice at 2E9 GC / mouse intramuscularly. Serum was collected at day 0 and periodically starting from day 14 post-AAV administration, and GLP-1 expression was assayed. At day 14, serum GLP-1 levels were increased, and starting from day 14, serum GLP-1 levels were remarkably stable for the duration of the study, which lasted 180 days (FIG. 1).

[0461] Conclusion

[0462] These results demonstrate the sustained, long-term expression following intramuscular administration of an AAV vector comprising a transgene.

[0463] Example 2: Delivery of Cre recombinase using lipid nanoparticles (LNPs) terminates expression of a transgene delivered with an AAV vector

[0464] Objective

[0465] The objective of this study was to terminate expression of a transgene delivered with an AAV vector using a Cre recombinase delivered using an LNP.

[0466] Materials and Methods

[0467] A vector construct was designed, AAV-GLP-1 -STOP, in which the transgene Glucagon-like peptide-1 (GLP-1 ) is flanked by the loxP sites. The construct also included a cytomegalovirus (cmv) promoter and a polyadenylation signal (pA), as shown in FIG. 2A.

[0468] An experiment was performed to measure the amount of secreted GLP-1 for 2-4 days post LNP delivery for two different conditions tested: negative control mRNA and Cre mRNA. The experiment was performed in vitro in 293 cells and the amount of GLP-1 production from the AAV-GLP-1 -STOP construct PATENT

[0469] ATTORNEY DOCKET NO.: 51772-016WO2 was measured. The amount of secreted GLP-1 (pM) was plotted against the day post LNP delivery. The steps performed on each day of the experiment are as follows:

[0470] D-1 (Day -1 ): Transduce 293s with AAV-GLP-1 -STOP, Multiplicity of Infection (MOI) = 1.2 E6GC / cell

[0471] DO (Day 0): Transfect with LNP mRNA

[0472] D1 (Day 1 ): Wash cells

[0473] D2 (Day 2): Sample supernatant

[0474] D3 (Day 3): Sample supernatant

[0475] D4 (Day 4): Sample supernatant

[0476] Results

[0477] In result, it was observed that Cre mRNA was able to reduce production of secreted GLP-1 compared to the negative control mRNA (FIG. 2B).

[0478] Example 3: Administration of Cre recombinase to terminate expression of a heterologous protein in a system-wide manner

[0479] This example illustrates that an intramuscularly administered Cre recombinase (such as LNP-Cre, a Cre recombinase administered using any other Cre delivery technique described herein, or a nucleic acid encoding Cre recombinase administered using any other Cre delivery technique described herein) can terminate the expression of a transgene of interest in a system-wide manner and throughout the body for a subject in which eliminating the expression presents a clinical benefit.

[0480] Using the compositions and methods described herein, the transgene expression cassette comprising a desired transgene (e.g., a therapeutic transgene encoding a therapeutic protein) and other functional components can be flanked by lox sites, thus generating a floxed transgene expression cassette. In some embodiments, the “floxed” transgene expression cassette is the one being genetically manipulated by the Cre-lox system. In some embodiments, the functional component is flanked by the lox sites, thus generating a “floxed” functional component. In some embodiments, the “floxed” functional component is the one being genetically manipulated by the Cre-lox system. In some embodiments, the functional component is a promoter, an enhancer, or a terminator, such as a pA signal. In some embodiments, the open reading frame of the transgene encoding the protein of interest is flanked by lox sites, thus generating a floxed transgene. In some embodiments, the “floxed” transgene is the one being genetically manipulated by the Cre-lox system. In some embodiments, the floxed transgene or floxed transgene expression cassette is delivered using a viral vector described in section 1(B). In some embodiments, the floxed transgene or floxed transgene expression cassette is delivered using a viral vector such as an AAV. In some embodiments, the floxed transgene or floxed transgene expression cassette is delivered using a transfection technique described in section 1(C). In some embodiments, a host cell may be modified to comprise a floxed transgene or floxed transgene expression cassette using any of the techniques described in section 1(D).

[0481] As a non-limiting example of the utility of the Cre-lox systems described herein, these systems may be applied to the treatment of a female subject with an inherited bleeding disorder that has PATENT

[0482] ATTORNEY DOCKET NO.: 51772-016WO2 previously undergone gene therapy to increase the expression of a circulating blood clotting protein in an effort to reduce the risk of hemorrhage. The gene therapy in this example included administration of a recombinant AAV vector comprising a transgene encoding the circulating protein.

[0483] After administration of the AAV comprising the floxed transgene or floxed transgene expression cassette encoding the circulating protein, the relative protein expression levels detected in a blood sample from the subject are determined to be in a normal range (e.g., an expression level within an accepted range). However, subsequent to treatment with this gene therapy agent, the subject is determined to have developed a chronic autoimmune disorder.

[0484] A skilled practitioner (e.g., a clinician) may determine that the patient’s new disease state and / or any associated therapeutic interventions are contraindicated for the originally administered transgene, posing the risk for an adverse event. To mitigate the risk of developing one or more adverse events, the subject may undergo treatment with a Cre recombinase (e.g., LNP-Cre, a Cre recombinase administered using any other Cre delivery technique described herein, or a nucleic acid encoding Cre recombinase administered using any other Cre delivery technique described herein) to reduce the expression of the transgene to a level beneath which the contraindication is obviated. In some embodiments, Cre recombinase is delivered using a viral vector described in section 1(B). In some embodiments, Cre recombinase is delivered using a viral vector such as an AAV. In some embodiments, Cre recombinase is delivered using a transfection technique described in section 1(C). In some embodiments, a host cell may be modified to comprise a transgene such as Cre recombinase using any of the techniques described in section 1(D). In some embodiments, Cre recombinase is delivered using an LNP described in section 11(D)

[0485] Upon administration, the Cre recombinase catalyzes site-specific recombination between the sequences flanked by the lox sites, thus reducing transgene expression. This reduction in transgene expression may be effectuated throughout the patient’s body (e.g., systemically) using a non-specific recombinase delivery technique described herein. Alternatively, transgene expression can be reduced within one or more specific cells, tissues, or organs of interest using a targeted recombinase delivery technique described herein, such as by direct administration of a recombinase (or nucleic acid encoding the same) to a desired location of the patient’s body or by administration of a recombinase that is operably linked to a transcription regulation element that is specifically active in one or more desired cell types or tissue types. In yet another example, the recombinase may be delivered by way of a viral vector having a tropism for one or more desired cells, tissues, or organs, thereby restricting recombinase expression (and consequent transgene reduction) to one or more specific regions of interest while preserving transgene expression elsewhere in the patient’s body. A specific example of tissue-specific transgene reduction is outlined in Example 3, below.

[0486] Example 4: Administration of Cre recombinase to terminate expression of a heterologous protein in a tissue-specific manner

[0487] This example illustrates a method of terminating the expression of a gene such as a transgene that encodes a polypeptide or a protein of interest or a fragment thereof (e.g., a therapeutic polypeptide or a therapeutic protein or fragment thereof) in a tissue-specific manner by administering to a subject a PATENT

[0488] ATTORNEY DOCKET NO.: 51772-016WO2 polynucleotide that comprises the floxed transgene (or floxed transgene expression cassette comprising a desired transgene (e.g., a therapeutic transgene encoding a therapeutic protein) and other functional components) to increase expression of the transgene and then administering to the subject a tissuespecific Cre recombinase to terminate the expression of the transgene in a specific tissue.

[0489] DNA modification can be carried out selectively in the specific cell type or tissue desired, for example, by placing the Cre enzyme under the control of a cell type- or tissue-specific promoter, such as a muscle specific promoter. Exemplary muscle-specific Cre promoters of the disclosure include, without limitation, ACTA1 (HSA), Ckmm (Mck), Myf5, Myh6 (aMHC), and Talgn (SM22a), as well as those described in Skopenkova, et al, 2021 . Acta Naturae 13:47-58. Other tissue-specific regulatory elements are known in the art and include, without limitation, the albumin promoter (liver specific; Pinkert, et al, 1987. Genes Dev. 1 : 268-277). In some embodiments, the muscle specific promoter is a phosphoglycerate kinase (PGK) promoter, a desmin promoter, a muscle creatine kinase promoter, a myosin light chain promoter, a myosin heavy chain promoter, a cardiac troponin C promoter, a troponin I promoter, a myoD gene family promoter, an actin alpha promoter, an actin beta promoter, an actin gamma promoter, a promoter within intron 1 of ocular paired like homeodomain 3, a cytomegalovirus promoter, or a chicken-p-actin promoter. In some embodiments, the Cre enzyme may be placed under the control of a muscle specific enhancer. In some embodiments, the muscle specific enhancer is a muscle creatine kinase enhancer, a distal regulatory element (DRE) enhancer, a smooth muscle myosin heavy chain (SMHC) enhancer, a dystrophin intron 1 enhancer, a MyoD enhancer, or a myocyte enhancer factor 2 (MEF2) enhancer.

[0490] In some embodiments, a cell type- or tissue-specific Cre recombinase is delivered using a viral vector described in section 1(B). In some embodiments, a cell type- or tissue-specific Cre recombinase is delivered using a viral vector such as an AAV. In some embodiments, a cell type- or tissue-specific Cre recombinase is delivered using a tissue-tropic viral vector (such as a muscle-tropic viral vector or AAV vector). In some embodiments, a cell type- or tissue-specific Cre recombinase is delivered using a transfection technique described in section 1(C). In some embodiments, a host cell may be modified to comprise a transgene such as a cell type- or tissue-specific Cre recombinase using any of the techniques described in section 1(D). In some embodiments, a cell type- or tissue-specific Cre recombinase is delivered using an LNP in section 11(D).

[0491] In this example, a subject (e.g., a human subject) having or at risk of developing a disease or a condition (e.g., a disease or a condition listed in Table 3) may be treated with a form of gene therapy that comprises intramuscularly administering to the subject an effective amount of a pharmaceutical composition comprising a polynucleotide that comprises a floxed transgene that encodes a therapeutic polypeptide or a therapeutic protein or a fragment thereof (or a floxed transgene expression cassette that comprises a transgene that encodes a therapeutic polypeptide or a therapeutic protein or a fragment thereof). In some embodiments, the open reading frame of the transgene encoding the therapeutic protein is flanked by lox sites, thus generating a floxed transgene. In some embodiments, the transgene expression cassette that comprises the open reading frame of the transgene encoding the therapeutic protein is flanked by lox sites, thus generating a floxed transgene expression cassette. The pharmaceutical composition may further comprise a delivery vehicle for effective delivery of the PATENT

[0492] ATTORNEY DOCKET NO.: 51772-016WO2 polynucleotide to the subject, in which the delivery vehicle is a vector such as a viral vector (e.g., an AAV vector (e.g., a recombinant AAV vector)), a liposome, or a microvesicle, among other suitable forms of delivery described herein. The pharmaceutical composition may further comprise components of a genetic engineering system (e.g., CRISPR or a transposon system) for delivering the polynucleotide to the subject. In some embodiments, the floxed transgene or floxed transgene expression cassette is delivered using a viral vector described in section 1(B). In some embodiments, the floxed transgene or floxed transgene expression cassette is delivered using a viral vector such as an AAV. In some embodiments, the floxed transgene or floxed transgene expression cassette is delivered using a transfection technique described in section 1(C). In some embodiments, a host cell may be modified to comprise a floxed transgene or floxed transgene expression cassette using any of the techniques described in section 1(D).

[0493] After administration of the original gene therapy agent to the patient, the patient may be evaluated to determine whether the expressed transgene is safe and effective for its intended purpose. In this example, the patient may develop an immunogenic reaction to the protein product encoded by the originally provided transgene, for instance, due to the fact that the patient does not express the transgene endogenously and, thus, has not been immunologically tolerized against the protein product. In this scenario, to avoid the potentially dangerous immunogenic reaction, the patient may be administered a recombinase (e.g., a Cre recombinase described herein) so as to terminate expression of the transgene (i.e., to reduce expression of the transgene to a level beneath which the risk of immunogenicity has been obviated). The administration of the recombinase may be by way of a single, saturating dosage that, alone, is sufficient to reduce expression of the transgene to a safe level. Alternatively, the recombinase may be administered in a plurality of serial doses, with intermittent evaluation of transgene expression levels, until it is determined that the patient’s transgene expression level has reached a safe range.

[0494] Example 5: Administration of a Cre recombinase to down-titrate expression of a transgene to within a desired therapeutic window

[0495] This example illustrates that a Cre recombinase (such as an intramuscularly administered LNP- Cre, a Cre recombinase administered using any other Cre delivery technique described herein, or a nucleic acid encoding Cre recombinase administered using any other Cre delivery technique described herein) can fine-tune the expression of a transgene of interest, either in a system-wide or a tissue-specific manner, as desired.

[0496] In this example, a female subject with a disorder that is amenable to treatment by expression of a protein of interest may be administered a transgene encoding the desired protein. The transgene may be provided, for instance, by way of any gene therapy vector described herein, such as by way of a recombinant AAV vector comprising the transgene. The transgene may be flanked by lox sites, as is described in the sections above. Alternatively, a component of the transgene expression cassette (e.g., a promoter or enhancer) may be flanked by lox sites, or the entire transgene expression cassette may be flanked by lox sites. PATENT

[0497] ATTORNEY DOCKET NO.: 51772-016WO2

[0498] Subsequent to the initial administration of the gene therapy agent, the patient may be evaluated to determine whether the expression level of the desired product encoded by the provided transgene is within a range that is simultaneously safe and effective for the disease being treated. In some embodiments, the patient may be determined to exhibit a transgene expression level following the initial administration that is higher than that which is necessary for the desired therapeutic effect to manifest. Additionally or alternatively, the patient may be determined to exhibit a transgene expression level following the initial administration that is higher than that which is safely tolerated.

[0499] In either of these circumstances, the patient may be administered a recombinase (e.g., a Cre recombinase) using any of the delivery modalities described herein, with the aim of down-titrating the expression level of the transgene to within a therapeutic window that is simultaneously safe and effective for the underlying disease being treated. For instance, the Cre recombinase may be delivered using a viral vector outlined in section 1(B). In some embodiments, the Cre recombinase is delivered using an AAV vector. In some embodiments, the Cre recombinase is delivered using a transfection technique described in section 1(C). In some embodiments, a host cell may be modified to comprise a transgene that encodes the Cre recombinase (e.g., using any of the techniques described in section 1(D)). In some embodiments, the Cre recombinase is delivered using an LNP, as is in section 11(D).

[0500] Upon administration, the Cre recombinase catalyzes site-specific recombination between the sequences flanked by the lox sites, thereby excising the transgene, a component of the transgene expression cassette, or the entire transgene expression cassette. This, in turn, reduces expression of the transgene in the patient.

[0501] Following administration of the recombinase to the patient, the patient may be evaluated to determine whether the expression level of the transgene has been sufficiently reduced to a level that is within a desired therapeutic window. This down-titration may be accomplished, for example, by way of a single, sub-saturating dosage of the recombinase that, alone, is sufficient to reduce expression of the transgene to within a range that is safe and effective. Alternatively, the recombinase may be administered by way of a series of repeat doses that, together, are sufficient to down-titrate expression of the transgene to within a therapeutically acceptable window. The patient may be monitored, as needed, following each dosage of the recombinase so as to determine the point at which the expression level of the original transgene has reached a level that is within the intended range.

[0502] The down-titration of transgene expression may be achieved either systemically or in a cell-, tissue-, or organ-specific manner. For instance, upon administration of the original transgene to the patient, it may be determined that the level of transgene expression is acceptable within one or more specific areas of the patient’s body, but is elevated in one or more other areas to beyond a threshold that is safe or necessary for the therapeutic phenotype to manifest. For instances in which systemic downtitration of transgene expression is desired, the recombinase may be provided to the patient in a manner that fosters recombinase expression non-specifically, such as by way of a nucleic acid that distributes approximately evenly throughout the patient’s body and / or by way of a DNA polynucleotide that is operably linked to a ubiquitous transcription regulatory element (e.g., a ubiquitous promoter or enhancer). For instances in which cell-, tissue, or organ-specific down-titration of transgene expression is desired, the recombinase may be provided to the patient in a manner that promotes recombinase expression PATENT

[0503] ATTORNEY DOCKET NO.: 51772-016WO2 asymmetrically in one or more desired regions, such as by way of a nucleic acid that distributes specifically to a target location (e.g., using a viral vector having a tropism specific for the desired location) and / or by way of a DNA polynucleotide that is operably linked to a transcription regulatory element having preferential activity in the desired cell, tissue, or organ.

[0504] Example 6: Administration of a Cre recombinase to down-titrate expression of a transgene in vivo Objective

[0505] The objective of this study was to terminate expression of an AAV-delivered transgene expressed in mice using a Cre recombinase delivered using an LNP-mRNA.

[0506] Materials and Methods

[0507] A vector construct was produced, AAV-GLP-1 -STOP, in which the genome contained a GLP-1 gene, where the majority of the coding region for GLP-1 was flanked by loxP sites. The construct also included a cmv promoter and a polyadenylation signal (pA), as shown in FIG. 3A. In addition to this test vector, a control AAV vector was produced, AAV-GLP-1 , containing a genome with the same elements but without the loxP sites. The AAV capsid for both vectors was AAV9.

[0508] Next, an LNP-Cre mRNA formulation was generated. The LNP was similar to covid vaccine LNP- mRNA preparations in terms of lipid composition. The LNP-mRNA was formulated at 0.25 mg / ml for delivery to the mice.

[0509] Results

[0510] AAV-GLP-1 -STOP and AAV-GLP-1 were delivered to identical cohorts of rag mice at 2E9 GC / mouse intramuscularly. Serum was collected at day 14 post-AAV administration, and GLP-1 expression was assayed. Nearly identical expression levels of the reporter GLP-1 from both AAV-GLP-1 - STOP and AAV-GLP-1 were observed, indicating that the introduction of the loxP sites did not interfere with baseline expression from the construct (FIG. 3B).

[0511] Also on day 14, cohorts of mice were dosed with 10 pl of either PBS (negative control) or LNP- Cre at the same injection site (rear leg) as the previous AAV dosing. On day 28, serum was collected again, and GLP-1 expression was measured. Treatment at the AAV-GLP-1 -STOP injection site with LNP- Cre caused a reduction in expression when compared to PBS-treated mice. This was not true for the AAV-GLP-1 -expressing mice, in which the expression cassette lacks loxP sites. This result is consistent with the specific action of Cre recombinase on AAV genomes to reduce therapeutic protein expression (FIG. 3C).

[0512] Serum GLP-1 expression for the AAV-GLP-1 -STOP mice was monitored for an additional 20 subsequent weeks. At days 70, 112, and 147, these mice were dosed with either PBS or LNP-Cre as before. In the weeks following each LNP-Cre dose, a drop in GLP-1 expression relative to the PBS- treated group was observed. The expression reduction was durable and accumulated through multiple doses of LNP-Cre (FIG. 3D). This demonstrates the 1 ) sustained expression-reduction of an AAV- expressed therapeutic transgene product in vivo and 2) the ability to iteratively tune down an appropriate PATENT

[0513] ATTORNEY DOCKET NO.: 51772-016WO2 therapeutic transgene expression in vivo through multiple sub-saturating doses of a compatible recombinase to the appropriate tissue.

[0514] Example 7: Dose-response of transgene expression in response to Cre recombinase administration

[0515] Objective

[0516] The objective of this study was to terminate expression of an AAV-delivered transgene expressed in mice using a Cre recombinase delivered using an LNP-mRNA and to further characterize the sensitivity of this system to the delivered concentration of Cre mRNA.

[0517] Materials and Methods

[0518] As described above, an AAV vector was produced, where the genome contained a GLP-1 gene, in which the majority of the coding region for GLP-1 was flanked by loxP sites (AAV-GLP-1 -STOP). The construct also included a cmv promoter and a pA signal, as shown in FIG. 3A. An LNP-Cre was produced as described above but at higher Cre mRNA concentrations, reaching a maximum concentration of 2.5 mg / ml.

[0519] Results

[0520] AAV-GLP-1 -STOP and AAV-GLP-1 were delivered to identical cohorts of rag mice at 2E8 GC / mouse intramuscularly. Serum was collected at day 46 post-AAV, and baseline GLP-1 expression was assayed for all mice. Also on day 46, cohorts of mice were dosed with 10 ul of either PBS (negative control) or LNP-Cre at the same injection site (rear leg) as the previous AAV dosing. One cohort of mice received PBS as a negative control, while the other cohorts of mice received LNP-Cre at one of three Cre mRNA concentrations (2.5 mg / ml, 1 mg / ml, and 0.5 mg / ml). On day 60, serum was collected again, and GLP-1 expression was measured. As before, treatment at the AAV-GLP-1 -STOP injection site with LNP- Cre caused a reduction in expression as compared to PBS. Higher Cre-mRNA concentrations were associated with more extensive therapeutic transgene expression reduction, demonstrating a path to achieve single-shot efficacy in the system (FIG. 4).

[0521] Example 8: Administration of a Cre recombinase to down-titrate expression of a transgene in vivo using alternate loxP sites incorporated into the therapeutic genome

[0522] Objective

[0523] The objective of this study was to terminate expression of an AAV-delivered transgene expressed in mice using a Cre recombinase delivered using an LNP-mRNA and to determine the suitability of alternate loxP sites to this transgene control system.

[0524] Materials and Methods

[0525] As described above, a vector construct was designed, AAV-GLP-1 -STOP, in which the transgene Glucagon-like peptide-1 (GLP-1 ) is flanked by the loxP sites. The construct also included a cmv promoter and a pA. Two similar vectors were produced, AAV-GLP-1 -STOP2 and AAV-GLP-1 -STOP3, in which the PATENT

[0526] ATTORNEY DOCKET NO.: 51772-016WO2 loxP sites had been exchanged for alternate lox variant sequences (FIG. 5A). LNP-Cre was produced as described above at 1 mg / ml Cre-mRNA.

[0527] Results

[0528] AAV-GLP-1 -STOP, AAV-GLP-1 -STOP2, and AAV-GLP-1 -STOP3 were delivered to identical cohorts of rag mice at 2E9 GC / mouse, intramuscularly. Serum was collected at day 28 post-AAV administration and baseline GLP-1 expression for all mice was assayed. Also on day 28, cohorts of mice were dosed with 10 ul of either PBS (negative control) or LNP-Cre at the same injection site (rear leg) as the previous AAV dosing. On day 42, serum was collected again, and GLP-1 expression was measured in all mice. Treatment of AAV-GLP-1 -STOP-, AAV-GLP-1 -STOP2-, and AAV-GLP-1 -STOP3-expressing mice with LNP-Cre caused a reduction in expression as compared to PBS (FIG. 5B), demonstrating the suitability of alternate loxP sites for application in the transgene expression control system.

[0529] Example 9: Administration of a Cre recombinase to down-titrate expression of a transgene in vivo using lox sites that flanking the pA element

[0530] Objective

[0531] The objective of this study was to determine the suitability of flanking the pA functional element with recombinase sites to modulate therapeutic protein expression from an AAV vector, as well as to determine the impact of recombinase site orientations on the efficacy of the expression control system.

[0532] While recombinases (e.g., Cre) can act on two recombinase sites (e.g., loxP sites) in a genome to cause a DNA recombination event, the result of the recombination depends on the orientation of the recombination sites. In the example of Cre recombinase, two inverted loxP sites (meaning that the forward-reading loxP site sequences occur on opposite strands of the DNA) typically result in an inversion of the intervening DNA by the Cre recombinase (FIG. 6A). In contrast, direct repeats of the loxP sites (meaning that the forward-reading loxP sites occur on the same strand of the DNA) typically result in the excision of the intervening DNA by the Cre recombinase (FIG. 6A). Since each of these outcomes (inversion and excision) may reduce expression from an AAV genome given the appropriate placement of lox sites, various combinations and orientations of lox sites were tested for their effect on AAV-mediated transgene expression.

[0533] The three elements that are most typically considered essential for protein expression cassettes are the promoter, which drives gene transcription; the open reading frame, which encodes the transgene of interest; and the pA signal, which serves to appropriately terminate an mRNA transcript and ensure its expression to protein in the cell. As demonstrated above, recombinase-catalyzed alterations of the open reading frame of the transgene of interest resulted in its separation from the promoter and terminator, thereby reducing expression of the protein of interest encoded by the transgene. Recombinase-catalyzed alterations to the pA signal were tested for their impact on AAV-encoded transgene expression.

[0534] Materials and Methods

[0535] Ten AAV vector constructs were designed encoding a promoter, therapeutic transgene (GLP-1 - Fc), and pA signal between the ITR packaging sequences. In each of these ten constructs, the pA signal PATENT

[0536] ATTORNEY DOCKET NO.: 51772-016WO2 sequence was flanked by recombinase sites (“floxed”). The constructs were distinguished from each other by the recombinase site sequence used (loxP, lox 71 , or Iox66) and / or the orientation of the sites with respect to the pA element (FIG. 6B). A control construct without recombinase sites was also produced. LNP-Cre was produced as described above at 1 mg / ml Cre-mRNA.

[0537] Results

[0538] AAV vectors encoding the control construct or one of the ten floxed pA constructs were produced. Each AAV vector was delivered to an identical cohort of rag mice at a dose of 5E8 GC / mouse intramuscularly. Serum was collected at day 30 post-AAV administration and baseline GLP-1 expression was assayed. Also on day 30, all mice were dosed with 10 pl of LNP-Cre at the same injection site (rear leg) as the previous AAV dosing. On day 42, serum was collected again, and GLP-1 expression was measured in all mice. The experimental timeline is shown in FIG. 6C.

[0539] In mice expressing the control construct, there was only a 16% reduction in GLP-1 expression following LNP-Cre administration on day 30 to day 42. On day 42, GLP-1 expression was sustained within an average of 84% of an initial expression level that was measured on day 30. In contrast, mice expressing the floxed pA constructs had a greater reduction in GLP-1 expression when compared to the control construct. In mice expressing the most responsive construct designs (PA2 and PA10), GLP-1 expression was reduced by about 2-fold in the 12 days following LNP-Cre delivery to the injection site (FIG. 6D). These data demonstrate the ability of both inversion and excision lox site orientations to control of AAV-delivered therapeutic transgene expression. The data also support floxed pA construct designs in the control of AAV-mediated transgene expression.

[0540] Other Embodiments

[0541] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

[0542] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations following, in general, the principles and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

[0543] Other embodiments are within the claims.

Claims

PATENTATTORNEY DOCKET NO.: 51772-016WO2What is claimed is:CLAIMS1 . A method of reducing expression or activity of a protein of interest in a subject in need thereof, the method comprising:(a) administering to the subject a recombinant nucleic acid comprising (i) a transgene expression cassette comprising a transgene encoding the protein of interest and (ii) a pair of recombinase recognition sites flanking the 5’ and 3’ ends of the expression cassette or any functional component thereof; and(b) subsequently administering to the subject a recombinase that specifically binds and cleaves at the recombinase recognition sites, thereby inactivating the transgene.

2. A method of reducing expression or activity of a protein of interest in a subject in need thereof, the method comprising administering to the subject a recombinase, wherein the subject has previously been administered a recombinant nucleic acid comprising (i) a transgene expression cassette comprising a transgene encoding the protein of interest and (ii) a pair of recombinase recognition sites flanking the 5’ and 3’ ends of the expression cassette or any functional component thereof, wherein the recombinase administered to the subject specifically binds and cleaves at the recombinase recognition sites, thereby inactivating the transgene.

3. A method of eliminating expression or activity of a protein of interest in an undesired tissue or organ of a subject while preserving expression or activity of the protein of interest in a desired tissue or organ of the subject, the method comprising:(a) administering to the subject a recombinant nucleic acid comprising (i) a transgene expression cassette comprising a transgene encoding the protein of interest and (ii) a pair of recombinase recognition sites flanking the 5’ and 3’ ends of the expression cassette or any functional component thereof, wherein the recombinant nucleic acid is administered to the subject systemically and / or wherein upon administration, the recombinant nucleic acid biodistributes to a plurality of tissues or organs; and(b) subsequently administering to the subject a recombinase that specifically binds and cleaves at the recombinase recognition sites, wherein the recombinase is specifically administered to and / or specifically biodistributes to the undesired tissue or organ, and is neither specifically administered to nor specifically biodistributes to the desired tissue or organ, thereby inactivating the transgene in the undesired tissue or organ while preserving the transgene in the desired tissue or organ.

4. A method of eliminating expression or activity of a protein of interest in an undesired tissue or organ of a subject while preserving expression or activity of the protein of interest in a desired tissue or organ of the subject, the method comprising administering to the subject a recombinase, wherein the subject has previously been administered a recombinant nucleic acid comprising (i) a transgene expression cassette comprising a transgene encoding the protein of interest and (ii) a pair of recombinase recognition sites flanking the 5’ and 3’ ends of the expression cassette or any functional componentPATENTATTORNEY DOCKET NO.: 51772-016WO2 thereof, wherein the recombinant nucleic acid has been administered to the subject systemically and / or wherein upon administration, the recombinant nucleic acid biodistributes to a plurality of tissues or organs, wherein the recombinase administered to the subject specifically binds and cleaves at the recombinase recognition sites, and wherein the recombinase is specifically administered to and / or specifically biodistributes to the undesired tissue or organ, and is neither specifically administered to nor specifically biodistributes to the desired tissue or organ, thereby inactivating the transgene in the undesired tissue or organ while preserving the transgene in the desired tissue or organ.

5. A method of reducing expression or activity of a protein of interest in a subject from a level that exceeds a therapeutic window for that protein in the subject to a level that is within a safe and effective range for the subject, the method comprising:(a) administering to the subject a recombinant nucleic acid comprising (i) a transgene expression cassette comprising a transgene encoding the protein of interest and (ii) a pair of recombinase recognition sites flanking the 5’ and 3’ ends of the expression cassette or any functional component thereof;(b) determining that the level of expression or activity of the protein of interest in the subject is higher than that which is safely tolerated by the subject; and(c) administering to the subject a sub-saturating amount of a corresponding recombinase that specifically binds the recombinase recognition sites, thereby reducing expression of the transgene.

6. A method of reducing expression or activity of a protein of interest in a subject from a level that exceeds a therapeutic window for that protein in the subject to a level that is within a safe and effective range for the subject, the method comprising administering to the subject a sub-saturating amount of a recombinase, wherein the subject has previously been administered a recombinant nucleic acid comprising (i) a transgene expression cassette comprising a transgene encoding the protein of interest and (ii) a pair of recombinase recognition sites flanking the 5’ and 3’ ends of the expression cassette or any functional component thereof, wherein following administration of the recombinant nucleic acid to the subject, the subject has been determined to exhibit a level of expression or activity of the protein of interest that is higher than that which is safely tolerated by the subject, and wherein the recombinase administered to the subject specifically binds the recombinase recognition sites, thereby reducing expression of the transgene.

7. The method of any one of claims 1 -6, wherein the pair of recombinase recognition sites flank the 5’ and 3’ ends of the expression cassette.

8. The method of any one of claims 1 -6, wherein the pair of recombinase recognition sites flank the 5’ and 3’ ends of the transgene.PATENTATTORNEY DOCKET NO.: 51772-016WO29. The method of any one of claims 1 -6, wherein the pair of recombinase recognition sites flank the 5’ and 3’ ends of any functional component of the expression cassette.

10. The method of claim 9, wherein the functional component is a promoter.11 . The method of claim 10, wherein the promoter is a muscle-specific promoter.

12. The method of claim 11 , wherein the muscle-specific promoter is a phosphoglycerate kinase(PGK) promoter, a desmin promoter, a muscle creatine kinase promoter, a myosin light chain promoter, a myosin heavy chain promoter, a cardiac troponin C promoter, a troponin I promoter, a myoD gene family promoter, an actin alpha promoter, an actin beta promoter, an actin gamma promoter, or a promoter within intron 1 of ocular paired like homeodomain 3, a cytomegalovirus promoter, or a chicken-p-actin promoter.

13. The method of claim 9, wherein the functional component is an enhancer.

14. The method of claim 13, wherein the enhancer is a muscle-specific enhancer.

15. The method of claim 14, wherein the muscle-specific enhancer is a muscle creatine kinase enhancer, a distal regulatory element (DRE) enhancer, a smooth muscle myosin heavy chain (SMHC) enhancer, a dystrophin intron 1 enhancer, a MyoD enhancer, or a myocyte enhancer factor 2 (MEF2) enhancer.

16. The method of claim 9, wherein the functional component is a terminator, and wherein the terminator is present within the transgene expression cassette in a reverse orientation relative to the orientation of the transgene.

17. The method of claim 16, wherein the terminator is a polyadenylation (pA) signal.

18. The method of any one of claims 1 -17, wherein the recombinant nucleic acid is administered to the subject in the form of a recombinant viral vector comprising the nucleic acid.

19. The method of claim 18, wherein the recombinant viral vector is a recombinant adeno-associated viral (AAV) vector.

20. The method of claim 19, wherein the recombinant AAV vector is a serotype 1 , 2, 3, 4, 5, 6, 7, 8, 9, rh10, or rh74 AAV vector, or wherein the AAV vector is encapsulated by one or more synthetic capsid proteins.PATENTATTORNEY DOCKET NO.: 51772-016WO221 . The method of any one of claims 1 -20, wherein expression of the transgene that encodes the protein of interest is sustained following administration of the recombinant nucleic acid to the subject.

22. The method of claim 21 , wherein expression of the transgene that encodes the protein of interest is sustained for between one week and ten years following administration, between 1 week and 7 years, between 1 week and 5 years, between 1 week and 3 years, between 1 week and 1 year, between 2 weeks and 24 weeks, or between 4 weeks and 6 weeks following administration.

23. The method of claim 21 , wherein expression of the transgene that encodes the protein of interest is sustained for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks, at least 16 weeks, at least 17 weeks, at least 18 weeks, at least 19 weeks, at least 20 weeks, at least 21 weeks, at least 22 weeks, at least 23 weeks, at least 24 weeks, at least 25 weeks, or at least 26 weeks following administration.

24. The method of any one of claims 1 -23, wherein expression of the transgene that encodes the protein of interest is sustained within fifty percent of an initial expression level.

25. The method of claim 24, wherein expression of the transgene that encodes the protein of interest is sustained within twenty percent of an initial expression level.

26. The method of claim 25, wherein expression of the transgene that encodes the protein of interest is sustained within ten percent of an initial expression level.

27. The method of any one of claims 1 -26, wherein the recombinase is administered to the subject in the form of a nucleic acid encoding the recombinase.

28. The method of claim 27, wherein the recombinase is administered to the subject by way of a viral vector comprising the nucleic acid encoding the recombinase.

29. The method of claim 28, wherein the viral vector comprising the nucleic acid encoding the recombinase is an AAV vector, optionally wherein the AAV vector is a serotype 1 , 2, 3, 4, 5, 6, 7, 8, 9, rh10, or rh74 AAV vector, or wherein the AAV vector is encapsulated by one or more synthetic capsid proteins.

30. The method of any one of claims 1 -26, wherein the recombinase is administered to the subject in the form of a lipid nanoparticle (LNP) comprising (i) the recombinase or (ii) a nucleic acid encoding the recombinase, optionally wherein the nucleic acid encoding the recombinase is an mRNA or DNA polynucleotide.PATENTATTORNEY DOCKET NO.: 51772-016WO231 . The method of any one of claims 1 -30, wherein the recombinase is administered to the subject in a tissue-specific or organ-specific manner.

32. The method of claim 31 , wherein the recombinase is administered directly to the subject’s muscle tissue or liver, optionally wherein the recombinase is administered to the subject intramuscularly.

33. The method of any one of claims 1 -32, wherein the recombinase is a Cre recombinase and the recombinase recognition sites are lox sites.

34. The method of claim 33, wherein the Cre recombinase has an amino acid sequence that is at least 85% identical (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of any one of SEQ ID NOs: 17-19.

35. The method of claim 33 or 34, wherein the lox sites comprise a pair of lox sites set forth in Table 2, herein.

36. The method of any one of claims 1 -35, wherein administration of the recombinase reduces expression of the protein of interest by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, as compared to expression of the protein of interest in the subject prior to administration of the recombinase.

37. The method of any one of claims 1 -36, wherein administration of the recombinase reduces the expression of the protein of interest by about 2-fold, by about 3-fold, by about 4-fold, by about 5-fold, by about 6-fold, by about 7-fold, by about 8-fold, by about 9-fold, by about 10-fold, by about 15-fold, by about 20-fold, by about 25-fold, by about 30-fold, by about 35-fold, by about 40-fold, by about 45-fold, by about 50-fold, or greater than 50-fold, as compared to expression of the protein of interest in the subject prior to administration of the recombinase.

38. The method of any one of claims 1 -37, wherein the recombinase is administered to the subject at least one week, one month, or one year following administration of the recombinant nucleic acid comprising the transgene encoding the protein of interest.

39. The method of claim 38, wherein the recombinase is administered at least one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, or 24 weeks following administration of the recombinant nucleic acid comprising the transgene encoding the protein of interest, optionally wherein the recombinase is administered between one week and ten years following administration of the recombinant nucleic acid comprising the transgene encoding the protein of interest.PATENTATTORNEY DOCKET NO.: 51772-016WO240. The method of claim 38, wherein the recombinase is administered to the subject from one month to one year following administration of the recombinant nucleic acid comprising the transgene encoding the protein of interest.41 . The method of claim 38, wherein the recombinase is administered to the subject from one year to ten years following administration of the recombinant nucleic acid comprising the transgene encoding the protein of interest.

42. The method of any one of claims 1 -41 , wherein the recombinase is administered to the subject in advance of the subject commencing treatment with a therapeutic intervention that is contraindicated with the protein of interest, optionally wherein the therapeutic intervention is to be chronically administered to the subject.

43. The method of claim 42, wherein the subject has had an immunogenic response against the protein of interest.

44. The method of claim 42, wherein the protein of interest accumulated to levels that have induced toxicity in the subject.

45. The method of claim 42, wherein the therapeutic intervention is an antiviral, antibacterial, or other microbial agent.

46. The method of claim 42, wherein the therapeutic intervention is a chemotherapy and / or immunotherapy regimen.

47. The method of claim 42, wherein the therapeutic intervention is one or more immunosuppressive agents.

48. The method of any one of claims 42-47, wherein the recombinase is administered to the subject from one day to 12 months prior to the subject receiving a therapeutic intervention that is contraindicated with the protein of interest, optionally wherein the therapeutic intervention is to be chronically administered to the subject.

49. The method of claim 48, wherein the recombinase is administered to the subject one week, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, 11 months, or 12 months prior to receiving a therapeutic intervention that is contraindicated with the protein of interest, optionally wherein the therapeutic intervention is to be chronically administered to the subject.PATENTATTORNEY DOCKET NO.: 51772-016WO250. The method of any one of claims 1 -49, wherein the recombinase is administered to the subject in a single dose.51 . The method of any one of claims 1 -49, wherein the recombinase is administered to the subject in a plurality of doses.

52. The method of claim 51 , wherein each dose is separated by at least one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, or ten weeks.

53. The method of any one of claims 1 -52, wherein the subject is a human.

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