Methods for reducing the expression or activity of a protein, for temporarily modulating the expression or activity of a protein, and for treating a disease.

Description

1 / 141 “METHODS FOR REDUCING THE EXPRESSION OR ACTIVITY OF A PROTEIN, FOR TEMPORARILY MODULATING THE EXPRESSION OR ACTIVITY OF A PROTEIN, AND FOR TREATING A DISEASE” TECHNICAL FIELD

[0001] This disclosure relates to methods of transient modulation of gene expression in a gene therapy context, as well as vectors, nucleic acids and related compositions that may be used in such methods. FUNDAMENTALS

[0002] 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 transgenic 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. During gene therapy with an AAV, it is sometimes desirable to decrease the potency of transgene expression or temporarily pause transgene expression for individuals requiring care due to a contraindication.There are limited options available to achieve adjustable transgene expression after administration with an AAV vector, often due to the risks of toxicity and unintended side effects of inhibitors, as well as incompatible biodistribution and pharmacokinetic profiles of an AAV and an inhibitor. There is still a need for mechanisms to achieve adjustable transgene expression after gene therapy with an AAV or other gene delivery vehicle. SUMMARY OF THE INVENTION

[0003] In one aspect, disclosure presents a method of Petition 870250098454, dated 10 / 28 / 2025, page 9 / 170 2 / 141 reduction of the expression or activity of a protein of interest in a subject, the method including: (a) administering to the subject a polynucleotide including (i) a transgene encoding the protein of interest and (ii) a binding site for an inhibitory RNA molecule; and subsequently; (b) administering to the subject an inhibitory RNA molecule, wherein the inhibitory RNA molecule is complementary to the binding site, and wherein the inhibitory RNA molecule is administered after a desired period of time following step (a); Optionally, the polynucleotide is administered to the subject intramuscularly. In some embodiments, the polynucleotide is administered in the form of a viral vector (e.g., a recombinant adeno-associated viral vector (AAV) containing the same). In some embodiments, the polynucleotide is administered in the form of a nonviral formulation, such as a liposome, microvesicle, CRISPR system, or transposon system, among others.

[0004] In another aspect, the disclosure presents a method for reducing the expression or activity of a protein of interest in a subject, the method including administering to the subject an inhibitory RNA molecule, wherein the subject has previously received a polynucleotide including (i) a transgene encoding the protein of interest and (ii) a binding site with complementarity to the inhibitory RNA molecule, optionally wherein the polynucleotide is administered to the subject intramuscularly. In some embodiments, the polynucleotide is administered in the form of a nonviral formulation, such as a liposome, microvesicle, CRISPR system, or transposon system, among others. [00 05] In another aspect, the disclosure presents a method for temporal modulation of the expression or activity of a protein of interest in Petition 870250098454, dated 10 / 28 / 2025, page 10 / 170 3 / 141 a subject, the method including: (a) administering to the subject a polynucleotide including (i) a transgene encoding the protein of interest and (ii) a binding site for an inhibitory RNA molecule, wherein administration of the polynucleotide increases the expression or activity of the protein of interest in a subject; and subsequently; (b) administering an inhibitory RNA molecule to the subject, wherein the inhibitory RNA molecule is complementary to the binding site, wherein the inhibitory RNA molecule is administered after a desired period of time following step (a), and wherein the administration of the inhibitory RNA molecule reduces the expression or activity of the protein of interest in a subject. In some embodiments, the polynucleotide is administered to the subject intramuscularly. In some embodiments, the polynucleotide is administered in the form of a nonviral formulation, such as a liposome, microvesicle, CRISPR system, or transposon system, among others.

[0006] In another aspect, the disclosure presents a method of treating a disease in a subject who needs it, the method including: (a) administering to the subject a polynucleotide including (i) a transgene encoding a protein of interest and (ii) a binding site for an inhibitory RNA molecule; and (b) administering to the subject an inhibitory RNA molecule, wherein the inhibitory RNA molecule is complementary to the binding site, and wherein the inhibitory RNA molecule is administered after a desired time period following step (a). In some embodiments, the polynucleotide is administered to the subject intramuscularly. In some embodiments, the polynucleotide is administered in the form of a nonviral formulation, such as a liposome, microvesicle, CRISPR system, or transposon system, among others. Petition 870250098454, dated 10 / 28 / 2025, page 11 / 170 4 / 141

[0007] In another aspect, the disclosure presents a method of treating a disease in a subject, the method including administering to the subject an inhibitory RNA molecule, wherein the subject has previously received a polynucleotide including (i) a transgene encoding the protein of interest and (ii) a binding site with complementarity to the inhibitory RNA molecule. In some embodiments, the polynucleotide is administered to the subject intramuscularly. In some embodiments, the polynucleotide is administered in the form of a nonviral formulation, such as a liposome, microvesicle, CRISPR system, or transposon system, among others.

[0008] In some embodiments of any of the foregoing aspects or embodiments of disclosure, the polynucleotide is administered to the subject in the form of a recombinant AAV containing the same.

[0009] In some embodiments, the recombinant AAV is administered to the subject between one and ten times (e.g., one, two, three, four, five, six, seven, eight, nine, or ten times). In some embodiments, the recombinant AAV is administered to the subject between one and five times (e.g., one, two, three, four, or five times). In some embodiments, the recombinant AAV is administered to the subject between three and five times (e.g., three, four, or five times). In some embodiments, the recombinant AAV is administered to the subject between one and three times (e.g., one, two, or three times). In some embodiments, the recombinant AAV is administered to the subject once.

[0010] In some modalities, the subject has or is at risk of developing a disease listed in Table 1.

[0011] In some forms, the transgene is a gene listed in Table 1.

[0012] In some modalities, the expression of the protein of interest is increased following administration of the recombinant AAV. In Petition 870250098454, dated 10 / 28 / 2025, p. 12 / 170 5 / 141 In some embodiments, the expression of the protein of interest is maintained for at least five days after administration of the recombinant AAV (e.g., from five days to ten years after administration of the recombinant AAV, such as from one month to five years after administration of the recombinant AAV, from one month to four years after administration of the recombinant AAV, from one month to three years after administration of the recombinant AAV, from one month to two years after administration of the recombinant AAV, or from one month to 12 months after administration of the recombinant AAV (e.g., 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 after administration of the recombinant AAV).

[0013] In some embodiments, the expression of the protein of interest is sustained for at least thirty days following administration of the recombinant AAV. In some embodiments, the expression of the protein of interest is sustained for at least sixty days following administration of the recombinant AAV. In some embodiments, the expression of the protein of interest is sustained for at least ninety days following administration of the recombinant AAV. In some embodiments, the expression of the protein of interest is sustained for at least 120 days following administration of the recombinant AAV.

[0014] In some embodiments, the expression of the protein of interest is sustained for at least one year following administration of the recombinant AAV. In some embodiments, the expression of the protein of interest is sustained for between one and five years following administration of the recombinant AAV. In some embodiments, the expression of the protein of interest is sustained for at least five years, six years, seven years, eight years, nine years, ten years, or more than ten years after administration of the AAV. Petition 870250098454, dated 10 / 28 / 2025, p. 13 / 170 6 / 141 recombinant.

[0015] In some embodiments, after administration of recombinant AAV to the subject, the protein of interest is expressed by the subject at a therapeutic level or at a subtherapeutic level.

[0016] In some embodiments, the inhibitory RNA molecule is selected from among a small interfering RNA (siRNA), an antisense oligonucleotide (ASO), a double-stranded RNA (dsRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), and a gapmer. In some embodiments, the inhibitory RNA molecule is an siRNA.

[0017] In some embodiments, the inhibitory RNA molecule also includes one or more modifications selected from among a nucleobase modification, a sugar modification, and an internucleoside linkage modification.

[0018] In some embodiments, the inhibitory RNA molecule also includes a hydrophobic moiety. In some embodiments, the hydrophobic moiety is cholesterol, among other hydrophobic substituents described herein.

[0019] In some embodiments, the inhibitory RNA molecule is formulated within a delivery vehicle. In some embodiments, the delivery vehicle is a lipid-based carrier, a liposome, or a lipid nanoparticle.

[0020] In some embodiments, the inhibitory RNA molecule lacks sufficient complementarity to hybridize with an endogenous RNA sequence that occurs naturally in a subject cell. In some embodiments, the inhibitory RNA molecule has less than 85% complementarity to an endogenous RNA sequence that occurs naturally in a subject cell (e.g., less than 80% complementarity to an endogenous RNA sequence that occurs Petition 870250098454, dated 10 / 28 / 2025, page 14 / 170 7 / 141 naturally occurring in a subject cell, less than 75% complementarity to an endogenous RNA sequence that occurs naturally in a subject cell, less than 70% complementarity to an endogenous RNA sequence that occurs naturally in a subject cell, less than 65% complementarity to an endogenous RNA sequence that occurs naturally in a subject cell, less than 60% complementarity to an endogenous RNA sequence that occurs naturally in a subject cell, less than 55% complementarity to an endogenous RNA sequence that occurs naturally in a subject cell, less than 50% complementarity to an endogenous RNA sequence that occurs naturally in a subject cell, less than 45% complementarity to an endogenous RNA sequence that occurs naturally in a subject cell, less than 40% complementarity to an endogenous RNA sequence that occurs naturally in a subject cell,less than 35% complementarity to an endogenous RNA sequence that occurs naturally in a subject cell, less than 30% complementarity to an endogenous RNA sequence that occurs naturally in a subject cell, less than 25% complementarity to an endogenous RNA sequence that occurs naturally in a subject cell, less than 20% complementarity to an endogenous RNA sequence that occurs naturally in a subject cell, less than 15% complementarity to an endogenous RNA sequence that occurs naturally in a subject cell, or less than 10% complementarity to an endogenous RNA sequence that occurs naturally in a subject cell).

[0021] In some embodiments, the inhibitory RNA molecule is complementary to an mRNA transcript or a portion thereof that is transcribed from the recombinant AAV vector, wherein the mRNA transcript encodes the protein of interest. Petition 870250098454, dated 10 / 28 / 2025, p. 15 / 170 8 / 141

[0022] In some embodiments, the inhibitory RNA molecule has at least 50% complementarity with the binding site of the inhibitory RNA molecule (for example, at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity) with the binding site of the inhibitory RNA molecule. In some embodiments, the inhibitory RNA molecule has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity with the binding site of the inhibitory RNA molecule.

[0023] In some embodiments, the inhibitory RNA molecule is 100% complementary to a sequence of at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 contiguous nucleotides within the binding site of the inhibitory RNA molecule.

[0024] In some embodiments, the binding site of the inhibitory RNA molecule is present in one or more of the following locations relative to the transgene: the 3' untranslated region (UTR), the 5' UTR, a region upstream of the start codon, a region downstream of the stop codon, or a region in the open reading frame of the transgene.

[0025] In some modalities, the inhibitory RNA molecule is administered intramuscularly to the subject, among other routes of administration described herein.

[0026] In some embodiments, administration of the inhibitory RNA molecule reduces the 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%, compared to the expression prior to administration of the inhibitory RNA molecule. In some embodiments, the Petition 870250098454, dated 10 / 28 / 2025, p. 16 / 170 9 / 141 Administration of the inhibitory RNA molecule reduces the expression of the protein of interest by approximately 2-fold, approximately 3-fold, approximately 4-fold, approximately 5-fold, approximately 6-fold, approximately 7-fold, approximately 8-fold, approximately 9-fold, approximately 10-fold, approximately 15-fold, approximately 20-fold, approximately 25-fold, approximately 30-fold, approximately 35-fold, approximately 40-fold, approximately 45-fold, approximately 50-fold or more than 50-fold, compared to the expression prior to administration of the inhibitory RNA molecule.

[0027] In some embodiments, administration of the inhibitory RNA molecule reduces expression of the protein of interest for at least five days. In some embodiments, administration of the inhibitory RNA molecule reduces expression of the protein of interest for at least thirty days. In some embodiments, administration of the inhibitory RNA molecule reduces expression of the protein of interest for at least sixty days. In some embodiments, administration of the inhibitory RNA molecule reduces expression of the protein of interest for at least ninety days. In some embodiments, administration of the inhibitory RNA molecule reduces expression of the protein of interest for at least 120 days.

[0028] In some embodiments, administration of the inhibitory RNA molecule reduces expression of the protein of interest for at least one year. In some embodiments, administration of the inhibitory RNA molecule reduces expression of the protein of interest for at least five years. In some embodiments, administration of the inhibitory RNA molecule reduces expression of the protein of interest for at least 5, 6, 7, 8, 9, 10 or more than 10 years.

[0029] In some forms, the subject is a human.

[0030] In another aspect, the disclosure presents a pharmaceutical composition including the polynucleotide of any of the previous embodiments and a pharmaceutically acceptable excipient, carrier or diluent. Petition 870250098454, dated 10 / 28 / 2025, p. 17 / 170 10 / 141 In some embodiments, the polynucleotide is incorporated into a viral vector (e.g., a recombinant AAV vector), a liposome, or a microvesicle. In some embodiments, the polynucleotide is provided as a component of a CRISPR system or as a component of a transposon system.

[0031] In some embodiments, the AAV vector is an AAV vector of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, rh10, or rh74. In some embodiments, the AAV vector is an AAV8 or AAV9 serotype vector. In some embodiments, the AAV vector contains one or more synthetic capsid proteins.

[0032] In another aspect, the disclosure presents a pharmaceutical composition including the inhibitory RNA molecule of any of the previous embodiments and a pharmaceutically acceptable excipient, carrier or diluent.

[0033] In another aspect, the disclosure presents a kit including a package insert that instructs the user to perform any of the previous methods. In some embodiments, the kit further includes the pharmaceutical composition including the inhibitory RNA molecule of any of the previous embodiments and a pharmaceutically acceptable excipient, carrier or diluent or the pharmaceutical composition including the polynucleotide of any of the previous embodiments and a pharmaceutically acceptable excipient, carrier or diluent. In some embodiments, the kit also includes one or more binding molecules to detect the expression or activity of the protein of interest. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The attached drawings are included to illustrate ways of carrying out the disclosure and to promote a further understanding of its implementations.

[0035] FIG. 1 is a diagram of an exemplary adeno-associated viral vector (AAV) transgene, showing inverted terminal repeats. Petition 870250098454, dated 10 / 28 / 2025, p. 18 / 170 11 / 141 (ITRs) of the AAV genome that flank the expression construct of the cyno dulaglutide (CyDula) transgene and a region in the 3' untranslated region (UTR) that serves as a target site for inhibitory small RNA molecules (siRNA).

[0036] FIG. 2 is a schematic of the experimental design for monitoring transgene expression levels in mice. On day 0 (d0), mice received an AAV9 vector containing the CyDula transgene intramuscularly. On day 13 (d13), a blood sample was obtained from the mice to establish baseline CyDula transgene expression levels. On day 14 (d14), mice received an appropriate siRNA or controls intramuscularly. Every two weeks after siRNA administration, additional blood samples were obtained to monitor possible changes in transgene expression levels over time.

[0037] FIG. 3A shows a graph plotting ELISA data showing serum protein concentrations measured by CyDula to assess potential adverse effects on gene expression after intramuscular administration of scrambled siRNA negative control molecules, in which one was modified to contain a hydrophobic fraction (C1-HM) and the other was not modified (C1).

[0038] FIG. 3B shows a graph plotting ELISA data showing relative serum CyDula protein concentrations compared to the mean serum expression level on day 13 for each group to assess potential adverse effects on gene expression after intramuscular administration of scrambled siRNA negative control molecules, in which one was modified to contain a hydrophobic fraction (C1-HM) and the other was not modified (C1).

[0039] FIG. 4 is a graph of ELISA data showing measured serum protein concentrations of CyDula after administration. Petition 870250098454, dated 10 / 28 / 2025, page 19 / 170 12 / 141 intramuscular injections of equal doses of sequence-equivalent siRNA molecules, in which one siRNA molecule was modified to contain a hydrophobic fraction (T1-HM) and the other was an unmodified siRNA (T1).

[0040] FIG. 5 is a graph depicting serum CyDula protein concentrations (relative to the mean serum CyDula expression level of the day 13 group) over time following intramuscular administration of 200 μg of hydrophobically modified T1-HM siRNA molecules, relative to a control injected with PBS.

[0041] FIG. 6 is a graph depicting serum CyDula protein concentrations (relative to the mean serum CyDula expression level of the day 13 group) over time following intramuscular administration of 50 μg of hydrophobically modified T1-HM siRNA molecules, relative to a PBS-injected control.

[0042] FIG. 7 is a graph depicting serum CyDula protein concentrations on day 28 relative to CyDula expression levels on day 13 after varying doses (6 μg, 12 μg, 25 μg, 50 μg, 100 μg, 200 μg, or 400 μg) of siRNA-1 relative to the same dose of a non-targeted siRNA.

[0043] FIG. 8 is a bar graph depicting serum protein concentrations of CyDula two weeks after intramuscular administration of the indicated siRNA molecules and doses relative to a control administered with PBS.

[0044] FIG. 9 is a bar graph depicting CyDula mRNA transcription levels in muscle tissue measured two weeks after intramuscular administration of the indicated siRNA molecules and doses relative to a control administered with PBS.

[0045] FIG. 10 is a dose-response curve showing the relative serum protein concentrations of CyDula two weeks after intramuscular administration of siRNA-1 at the indicated doses, compared Petition 870250098454, dated 10 / 28 / 2025, page 20 / 170 13 / 141 with the relative serum concentrations prior to siRNA administration.

[0046] FIG. 11 is a dose-response curve showing the levels of CyDula mRNA transcription in muscle tissue measured two weeks after intramuscular administration of siRNA-1 at the indicated doses, compared with the relative serum concentrations before siRNA administration. DEFINITIONS

[0047] Unless otherwise defined in this document, the scientific and technical terms used in this document have the meanings that are commonly understood by those skilled in the art. In case of any latent ambiguity, the definitions given in this document take precedence over any dictionary or extrinsic definition. Unless otherwise required by the context, singular terms should include plurals and plural terms should include singulars. The use of or means and / or, unless otherwise indicated. The use of the term including, as well as other forms such as includes and included, is not limiting.

[0048] As used herein, the term about, when applied to one or more values ​​of interest, refers to a value that is within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in any direction (greater or less than) of a stated reference value, unless otherwise indicated or evident from the context (except where such a number exceeds 100% of a possible value).

[0049] As used herein, the term “comprise” or variations thereof (e.g., comprehends or comprehending) shall be understood as including a feature or a plurality of features, but not excluding additional features or a plurality of features.

[0050] As used here in the context of a therapeutic intervention and a protein of interest, the term “contraindicated” refers to Petition 870250098454, dated 10 / 28 / 2025, page 21 / 170 14 / 141 cases in which the administration of the therapeutic intervention (e.g., a small molecule, biological product, cell therapy, gene therapy, surgical procedure, dietary supplement, or other therapeutic agent intended to produce a beneficial effect in a patient) would negate the effect or produce toxicity as a result of the expression of the protein of interest. Exemplary contraindications that may be encountered by a patient undergoing treatment with the compositions and methods of disclosure are described herein. In such cases, a patient may receive an inhibitory RNA molecule to temporarily suppress the expression of the protein of interest, at least until the therapeutic intervention has ceased.

[0051] As used herein, the terms “polynucleotide” or “nucleic acid” refer to polymers of nucleotides of any length and include DNA and / or RNA. 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 example, polynucleotides as defined herein include, without limitation, single-stranded and double-stranded DNA, DNA including single-stranded and double-stranded regions, single-stranded and double-stranded RNA and RNA including single-stranded and double-stranded regions, hybrid molecules comprising DNA and RNA which may be single-stranded or, more typically, double-stranded or include single-stranded and double-stranded regions. In addition, the term “polynucleotide,” as used herein, refers to triple-stranded regions comprising RNA or DNA or RNA and DNA.The strands in these regions can be of the same molecule or of different molecules. The regions may include all or more molecules, but usually involve only a region of some of the molecules. One of the molecules in a triple helix region is usually an oligonucleotide. The term "polynucleotide" specifically includes complementary DNA (cDNA). Petition 870250098454, dated 10 / 28 / 2025, page 22 / 170 15 / 141

[0052] A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogues. If present, the modification in the nucleotide structure may be conferred before or after the assembly of the polymer. The nucleotide sequence may be interrupted by non-nucleotide components. A polynucleotide may also be modified after polymerization, such as by conjugation with a labeling component.Other types of modifications include, for example, caps, substitution of one or more naturally occurring nucleotides by an analog, internucleotide modifications, such as, for example, those with uncharged bonds (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates and the like) and with charged bonds (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 chelating agents (e.g., metals, radioactive metals, boron, oxidative metals and the like), those containing alkylators, those with modified bonds (e.g., alpha anomeric nucleic acids), as well as unmodified forms of the polynucleotide(s).Furthermore, any of the hydroxyl groups normally present in sugars can be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or it can be conjugated to solid or semi-solid supports. The terminal 5' and 3' OHs can be phosphorylated or replaced by amines or organic capping groups of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized into 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'-O-methyl-, 2'-O-allyl-, 2'-fluoro-, or 2'-azido. Petition 870250098454, dated 10 / 28 / 2025, page 23 / 170 16 / 141 ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xyloses or lycoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester bonds may be replaced by alternative linking groups. These alternative linkage groups include, but are not limited to, embodiments in which the 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), where each R or R' is independently H or alkyl substituted or unsubstituted (1-20 C) optionally containing an ether (-O-), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl linkage. Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

[0053] As used herein, the terms “adenine” and “adenosine” are interchangeable terms with reference to a nucleotide having an adenine base. As used herein, the terms “cytosine” and “cytidine” are interchangeable terms with reference to a nucleotide having a cytosine base. As used herein, the terms “guanine” and “guanidine” are interchangeable terms with reference to a nucleotide having a guanine base. As used herein, the terms “thymine” and “thymidine” are interchangeable terms with reference to a nucleotide having a thymine base. As used herein, the terms “uracil” and “uridine” are interchangeable terms with reference to a nucleotide having a uracil base.

[0054] As used in this document, the term nucleoside refers to a molecule composed of a heterocyclic base and its sugar.

[0055] As used in this document, the term nucleotide refers to a nucleoside having a phosphate group, or a variant thereof, at its 3' or 5' sugar hydroxyl group. Examples of phosphate group variants Petition 870250098454, dated 10 / 28 / 2025, page 24 / 170 17 / 141 includes, but is not limited to, saturated alkyl phosphonates, unsaturated alkenyl phosphonates, phosphorothioates, and phosphoramidites.

[0056] 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 internucleosidic bonds (major structure), as well as oligonucleotides having non-naturally occurring (e.g., modified) portions that function similarly. These modified or substituted oligonucleotides are often preferred to native forms due to desirable properties such as greater cellular uptake, higher affinity for the target nucleic acid, and greater stability in the presence of nucleases.

[0057] 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 that begins with a start codon (e.g., methionine (ATG or AUG)) and ends with a stop codon (e.g., TAA, TAG, or TGA, or UAA, UAG, or UGA). An ORF typically codes for a protein. It is understood that the sequences disclosed herein may include additional elements, e.g., 5' and 3' UTRs, but that these elements, unlike the ORF, do not necessarily need to be present in an RNA polynucleotide (e.g., an mRNA transcript) disclosed herein.

[0058] As used herein, “messenger RNA”, “mRNA” or “mRNA transcript” is any RNA molecule that encodes a (at least one) protein (e.g., polypeptide) or fragment thereof and can be translated to produce the encoded protein or fragment thereof in vitro, in vivo, in situ or ex vivo. Structural and topological features, as well as Petition 870250098454, dated 10 / 28 / 2025, page 25 / 170 18 / 141 post-transcriptional modifications of mRNA are described here and are well known in the art.

[0059] As used herein, the term “inhibitory nucleic acid” refers to a nucleic acid comprising a guide strand sequence that hybridizes with at least a portion of a target nucleic acid, for example, ATXN2 RNA, mRNA, pre-mRNA, or mature mRNA, and inhibits its expression or activity. An inhibitory nucleic acid may target a protein-coding region (e.g., exon) or a non-coding region (e.g., 5'UTR, 3'UTR, intron, etc.) of a target nucleic acid. In some embodiments, the inhibitory nucleic acid is a single-stranded or double-stranded molecule. An inhibitory nucleic acid may further comprise a passenger strand sequence on a separate strand (e.g., double-stranded duplex) or on the same strand (e.g., single-stranded duplex structure and self-annealing).In some embodiments, an inhibitory nucleic acid is an interfering RNA molecule, such as short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), antisense oligonucleotide (ASO), or gapmer.

[0060] As used herein, the term siRNA refers to small interfering RNA duplexes that induce the RNA interference (RNAi) pathway. siRNA molecules can vary in length (generally between 10 and 30 base pairs) and can contain varying degrees of complementarity to their target mRNA. The term siRNA includes duplexes of two separate strands as well as single strands that optionally form hairpin structures including a duplex region.

[0061] As used in this document, the term antisense strand refers to the strand of the siRNA duplex that contains some degree of complementarity with the target gene.

[0062] As used in this document, the term sense tape refers to the siRNA duplex tape that contains complementarity with the tape. Petition 870250098454, dated 10 / 28 / 2025, p. 26 / 170 19 / 141 antisense.

[0063] The term “interfering RNA molecule” refers to an RNA molecule, such as a small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), or an antisense oligonucleotide (ASO) that suppresses the endogenous function of a target RNA transcript.

[0064] 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 this 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 pairing, while each unpaired nucleotide, and each incorrectly paired nucleotide, is referred to as a mismatch. Alignment for purposes of determining the percentage complementarity of the nucleic acid sequence can be achieved in various ways that are within the capabilities of one skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software.

[0065] As used herein, the term percentage (%) of 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 sequence alignment and the introduction of gaps, if necessary, to achieve the maximum percentage of sequence complementarity. A given nucleotide is considered complementary to a reference nucleotide, as described in this document, if the two nucleotides form Watson base pairs. Petition 870250098454, dated 10 / 28 / 2025, page 27 / 170 20 / 141 Canonical Crick base pairs. For the avoidance of doubt, Watson-Crick base pairs in the context of this 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 pairing, while each unpaired nucleotide, and each incorrectly paired nucleotide, is referred to as a mismatch. Alignment for the purpose of determining the percentage complementarity of nucleic acid sequences can be achieved in several ways that are within the capabilities of one skilled 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 sequence alignment, including any algorithms necessary to achieve maximum complementarity along the entire length of the sequences being compared.By way of illustration, the percentage complementarity of a given nucleic acid sequence, A, to a given nucleic acid sequence, B, (which may alternatively be expressed as a given nucleic acid sequence, A, having a certain percentage of complementarity to a given nucleic acid sequence, B) is calculated as follows: 100 multiplied by (the fraction X / Y) where X is the number of complementary base pairs in an alignment (e.g., as performed 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 percentage of sequence complementarity from A to B will not be equal to the percentage of sequence complementarity from B to A. As used in this document, a nucleic acid sequence of Petition 870250098454, dated 10 / 28 / 2025, p. 28 / 170 A 21 / 141 query is considered completely complementary to a reference nucleic acid sequence if the query nucleic acid sequence has 100% sequence complementarity with the reference nucleic acid sequence.

[0066] As used herein, a peptide is less than or equal to 50 amino acids in length, for example, approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0067] 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 have 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 in length. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogues of the foregoing. A polypeptide may be a single molecule or it may be a multi-molecular complex, such as a dimer, a trimer, or a tetramer.The term polypeptide can also be applied to polymers of amino acids in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.

[0068] As used herein, the term “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 have no functional activity. Functional proteins of this invention include, but are not limited to, enzymes, dehydrogenases, hydrolases, oxidoreductases, transferases, lyases, ligases, receptors, receptor ligands, cytokines, antibodies, molecules Petition 870250098454, dated 10 / 28 / 2025, page 29 / 170 22 / 141 Immunomodulatory enzymes are signaling molecules or proteins that are labeled 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 that 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 of viruses and infectious organisms, oncogenic products, thrombopoietin, erythropoietin, tissue plasminogen activator and any other biologically active protein that is desired for use in a clinical setting. Such proteins are well known in the art.Also included are deletion mutants of these proteins (for example, in which an individual residue or a plurality of residues are deleted from the sequence), individual domains of these proteins, fusion proteins made from these proteins, and mixtures of these proteins.

[0069] As used herein, the term “antibody” refers to a molecule that specifically binds to, or is immunologically reactive to, a particular antigen and includes at least the variable domain of a heavy chain, and typically includes at least the variable domains of both a heavy chain and 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 antibodies, Petition 870250098454, dated 10 / 28 / 2025, page 30 / 170 23 / 141 humanized, primatized or chimeric, single-stranded antibodies, epitope-binding fragments, for example, Fab, Fab' and F(ab')2, Fd, Fvs, single-stranded Fvs (scFv), single-stranded antibodies, disulfide-linked Fvs (sdFv), fragments comprising a Vl or Vh domain, fragments produced by a Fab expression library and anti-idiotypic antibodies (anti-Id). Antibody molecules of the invention may be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of the immunoglobulin molecule. Furthermore, unless otherwise indicated, the term monoclonal antibody (mAb) is intended to include intact molecules as well as antibody fragments (such as 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.

[0070] The term “antigen-binding fragment,” as used herein, refers to one or more fragments of an immunoglobulin that retain the ability to bind specifically to a target antigen. The antigen-binding function of an immunoglobulin can be performed by fragments of a full-length antibody. Antibody fragments can be a Fab, F(ab')2, scFv, SMIP, diabody, a triabody, an afibody, 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) an F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the Vh and Ch1 domains; (iv) an 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... Petition 870250098454, dated 10 / 28 / 2025, page 31 / 170 24 / 141 consists of a Vh domain; (vii) a dAb consisting of a Vh or Vl domain; (viii) an isolated complementarity-determining region (CDR); and (ix) a combination of two or more isolated CDRs that may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, Vl and Vh, are encoded by separate genes, they can be joined, using recombinant methods, by a linker that allows them to be made as a single protein chain in which the Vl and Vh regions pair up to form monovalent molecules (known as single-chain Fv (scFv)). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments can be researched for usefulness in the same way 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.

[0071] As used herein, percentage (%) of 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 sequence alignment and introduction of gaps, if necessary, to achieve the maximum percentage of sequence identity. Alignment for the purpose of determining the percentage of nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of a person skilled 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 sequence alignment, including any algorithms. Petition 870250098454, dated 10 / 28 / 2025, p. 32 / 170 25 / 141 are needed to achieve maximum alignment over the total length of the sequences being compared. For example, percentage sequence identity values ​​can be generated using the BLAST sequence comparison computer program.By way of illustration, the percentage of 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 may alternatively be expressed as a given nucleic acid or amino acid sequence, A that has a certain percentage of sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows: 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 percentage of sequence identity from A to B will not be equal to the percentage of sequence identity from B to A.

[0072] As used in this document, the term vector includes a nucleic acid vector, for example, a DNA vector such as a plasmid, an RNA vector, a 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, for example, WO1994 / 011026; incorporated herein by reference with regard to vectors suitable for the expression of a gene of interest. The expression vectors suitable for use with the compositions and methods described in this Petition 870250098454, dated 10 / 28 / 2025, p. 33 / 170 26 / 141 document contain a polynucleotide sequence, as well as, for example, additional sequence elements used for protein expression 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 in this document include plasmids containing regulatory sequences, such as promoter and enhancer regions, that direct gene transcription. Other vectors useful for the expression of a transgene contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of the mRNA resulting from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, an internal ribosomal entry site (IRES), and a polyadenylation signal site to direct efficient transcription of the gene carried in the expression vector.The expression vectors suitable for use with the compositions and methods described in this document may also contain a polynucleotide encoding a marker for selecting cells containing such a vector. Examples of a suitable marker are genes encoding antibiotic resistance, such as ampicillin, chloramphenicol, kanamycin, nourseotricin, or zeocin.

[0073] 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, caprine AAV, shrimp AAV, and any other AAV currently known or subsequently discovered. See, for example, Fields et al. Virology, 4th ed. Lippincott-Raven Publishers, Philadelphia, 1996. Additional serotypes and clades of AAV have been identified recently. (See, for example, Gao et al. J. Virol. 78:6381 (2004); Moris et al. Virol. 33:375 (2004). The genomic sequences of Petition 870250098454, dated 10 / 28 / 2025, p. 34 / 170 27 / 141 Several AAV serotypes, as well as the sequences of native inverted terminal repeats (ITRs), Rep proteins, and capsid subunits are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. See, for example, 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; whose publications are incorporated by reference herein for teaching nucleic acid and amino acid sequences in AAV. See also, for example, 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. Process. Nat. Acad. Science. 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; whose disclosures are incorporated by reference herein for the teaching of nucleic acid and amino acid sequences of AAV.

[0074] From a structural perspective, AAV is a non-pathogenic parvovirus composed of a 4.7 kb single-stranded DNA genome within a non-enveloped icosahedral capsid. The genome contains three open reading frames (ORFs) flanked by inverted terminal repeats (ITRs) that function as the viral origin of replication and packaging signal. The Rep ORF encodes four non-structural proteins that play roles in viral replication, transcriptional regulation, site-specific integration, and assembly. Petition 870250098454, dated 10 / 28 / 2025, page 35 / 170 28 / 141 of the virion. 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 alternative reading frame within the Cap gene produces the activator assembly protein (AAP), a viral protein that localizes AAV capsid proteins to the nucleolus and functions in the capsid assembly process. There are several natural (wild-type) serotypes and over 100 known variants of AAV, each of which differs in amino acid sequence, particularly in the hypervariable regions of the capsid proteins and therefore in their gene distribution properties. No AAV has been associated with any human disease, making recombinant AAV attractive for clinical applications.

[0075] Genomic sequences of several AAV serotypes, as well as sequences of native terminal repeats (TRs), Rep proteins, and capsid subunits, are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. See, for example, GenBank accession numbers NC_002077.1 (AAV1), AF063497. 1 (AAV1), NC_001401. 2 (AAV2), AF043303. 1 (AAV2), J01901. 1 (AAV2), U48704. 1 (AAV3A), NC_001729. 1 (AAV3A), AF028705. 1 (AAV3B), NC. 001829.1 (AAV4), U89790. 1 (AAV4), NC_006152. 1 (AA5), AF085716. 1 (AAV5), 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 (AAVrh10); whose disclosures are incorporated by reference herein for the teaching of AAV nucleic acid and amino acid sequences. See also, for example, Srivistava et al. (1983) J. Virologia 45:555; Chiorini et al. (1998) J. Virologia 71:6823; Chiorini et al. (1999) J. Virologia 73:1309; BantelSchaal et al. (1999) J. Virologia 73:939; Xiao et al. (1999) J. Virologia 73:3994; Muramatsu et al. (1996) Virologia 221:208; Sombra et al. (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Ciência. USA 99: 11854; Moris et al. (2004) Petition 870250098454, dated 10 / 28 / 2025, p. 36 / 170 29 / 141 Virology 33: 375-383; international patent publications WO 00 / 28061, WO 99 / 61601, WO 98 / 11244; and U.S. Patent No. 6,156,303.

[0076] As used herein, the term “transgene” refers to a recombinant nucleic acid (e.g., DNA or cDNA) that encodes a gene product (e.g., a therapeutic gene product). The gene product may be an RNA, peptide, polypeptide, or protein. In addition to the coding region for the gene product, the transgene may include or be operationally linked to one or more elements to facilitate or enhance expression, such as a promoter, enhancers, destabilizing domains, response elements, reporter elements, insulator elements, polyadenylation signals, and / or other functional elements. The modalities may utilize any suitable promoter, enhancers, destabilizing domains, response elements, reporter elements, insulator elements, polyadenylation signals, and / or other functional elements.

[0077] As used herein, the terms “wild type,” “natural occurrence,” or “non-mutant” form of a gene refer to a nucleic acid that codes for a protein associated with normal or non-pathogenic activity (e.g., a protein without mutation). In some embodiments, the wild-type gene may serve as a reference for comparison to a variant gene that is associated with a genetic disorder, for example, as described in Table 1 herein.

[0078] As used herein, the term “variant” or “mutant” refers to any gene with an alteration 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 that includes a single nucleotide point mutation resulting in a premature stop codon, a single nucleotide insertion, a single nucleotide deletion, the insertion of two or more contiguous nucleotides, the deletion of two or Petition 870250098454, dated 10 / 28 / 2025, page 37 / 170 30 / 141 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 can occur in any region of the gene. Genetic 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 repeated expansions.

[0079] As used herein, the terms “hybridization,” “annealing,” or “linking” of nucleic acids are obtained when one or more nucleoside residues within a polynucleotide base pair with one or more complementary nucleosides to form a stable duplex. Base pairing is typically driven by hydrogen bonding events. Hybridization includes Watson-Crick base pairs formed from natural and / or modified nucleobases. 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 do not need to be 100% complementary to undergo hybridization.For example, a nucleic acid may be, for instance, 95% complementary, 90% complementary, 85% complementary, 80% complementary, 75% complementary, 70% complementary, 65% complementary, 60% complementary, 55% complementary, 50% complementary, or less, with respect to another nucleic acid, but the two nucleic acids may still form sufficient base pairs with each other to hybridize.

[0080] As used herein, the phrase operably linked refers to a functional link between two or more similar molecules, constructs, transcripts, entities, or portions. For example, a DNA segment can be operatively linked to another DNA segment if they are positioned relative to each other on the same DNA molecule. Petition 870250098454, dated 10 / 28 / 2025, p. 38 / 170 31 / 141 contiguous and have a structural or functional relationship, such as a promoter or enhancer that is positioned relative to a coding region in order to facilitate transcription of the coding region. In other examples, operatively linked nucleic acids are not contiguous, but are positioned in such a way that they have a functional relationship to each other as nucleic acids or as proteins that are expressed by them. Enhancers, for example, do not need to be contiguous. Ligation can be achieved by ligation at convenient restriction sites or by the use of synthetic oligonucleotide adapters or ligands.

[0081] As used herein, the term “contact” (i.e., bringing a cell into contact 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 so that the agent and the subject’s cells are brought into contact in vivo. The term “contact” 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).

[0082] As used herein, the terms associated with, conjugated, linked, attached, and fastened, when used with respect to two or more portions, mean that the portions are physically associated or linked to each other, either directly or through one or more additional portions that serve as a linking agent, to form a structure that is sufficiently stable that the portions remain physically associated under the conditions in which the structure is used, for example, physiological conditions. An association need not be strictly by means of direct covalent chemical bonding. It may also suggest ionic or hydrogen bonding or a connectivity based on hybridization sufficiently stable such that the associated entities remain physically associated. Petition 870250098454, dated 10 / 28 / 2025, p. 39 / 170 32 / 141

[0083] As used herein, “modified” refers to an altered 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 can 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 DNA or 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.

[0084] As used in this document, the term expressed 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.The expression of a gene of interest in a subject may manifest itself, for example, by the detection of: an increase in the amount or concentration of mRNA encoding a corresponding protein (as assessed, for example, 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 amount or concentration of a corresponding protein (as assessed, for example, using protein detection methods described herein or known in the art, such as enzyme-linked immunosorbent assays (ELISA), Western blot, or mass spectrometry). Petition 870250098454, dated 10 / 28 / 2025, page 40 / 170 33 / 141 among others) and / or an increase in the activity of a corresponding protein (for example, in the case of an enzyme, as assessed using an enzyme activity assay known in the art) in a sample obtained from the subject.

[0085] 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 between 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 subset of cells, or in a plate or receptacle. In some embodiments, stability refers to the half-life of the biological material or molecule.

[0086] As used herein, the term “reduce,” with respect to gene expression (i.e., expression levels), refers to the decrease (e.g., suppression, reduction, minimization, diminution, or dampening) of the rate, degree, or magnitude of any one or more aspects of gene expression (e.g., transcription, RNA processing, translation, post-translational modifications, and / or protein activity (e.g., protein function or catalytic activity)). The reduction of gene expression (e.g., expression of a transgene encoding a protein of interest; e.g., a transgene in a recombinant AAV vector, as described herein) may be induced by an exogenous agent, such as an inhibitory RNA, as described herein.The degree and / or duration of time during which gene expression is reduced may depend on the physicochemical properties of one or more inhibitory RNA molecules, including the physicochemical properties of a delivery vehicle, the frequency of delivery, the route of delivery, and / or the amount administered. Gene expression levels may be reduced by approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more. Petition 870250098454, dated 10 / 28 / 2025, p. 41 / 170 34 / 141 100% relative to the expression levels of one or more reference samples, such as, for example, a sample obtained before the administration of an inhibitory RNA molecule. The reduction in gene expression can be confirmed or quantified by any suitable method for measuring the expression of a nucleic acid or protein (including protein activity) known in the art or described herein, in which a sample in which gene expression is reduced is compared to an equivalent reference sample (e.g., a sample obtained before the administration of an inhibitory RNA molecule).

[0087] As described in this document, the term exogenous describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is not found in nature in a particular organism (e.g., a human being) 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 supplied from a source external to an organism or to cultured matter extracted from it.

[0088] As used in this document, the term “in vitro” refers to events that occur in an artificial environment, for example, in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than inside an organism (e.g., animal, plant, or microbe).

[0089] As used in this document, the term “in vivo” refers to events that occur within an organism (e.g., animal, plant, or microbe, or a cell or tissue thereof).

[0090] 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 removed from its natural environment (by Petition 870250098454, dated 10 / 28 / 2025, page 42 / 170 35 / 141 example, from the body or natural structure) and placed in an artificial environment (e.g., a test tube or culture plate, flask, or other receptacle) for experimental or clinical applications. In some cases, ex vivo experimentation or applications may involve the administration (e.g., implantation, injection, depot, infusion, among other suitable routes of administration) of the component to the same subject or to a separate recipient subject after one or more ex vivo applications.

[0091] As used in this document, treatment and treating, in reference to a disease or condition, refer to an approach to achieving beneficial or desired outcomes, for example, clinical outcomes. Beneficial or desired outcomes may include, but are not limited to, relief or improvement of one or more symptoms or conditions; reduction in the extent of the disease or condition; stabilization of the disease, disorder, or condition (i.e., without worsening); prevention of the spread of diseases or conditions; delaying or slowing the progression of the disease or condition; improvement or palliation of the disease or condition; and detectable or undetectable remission (partial or total). “Improvement” or “palliation” of a disease or condition means that the extent and / or undesirable clinical manifestations or symptoms of the disease, disorder, or condition are reduced and / or the time course of progression is slowed or prolonged, compared to the extent or time course in the absence of treatment.“Treatment” can also mean prolonging survival compared to the expected survival without treatment. Those who need treatment include those who already have the condition or disorder, as well as those likely to have or at risk of having the condition or disorder (for example, for prophylactic treatment).

[0092] 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, Petition 870250098454, dated 10 / 28 / 2025, page 43 / 170 36 / 141 plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, bronchoalveolar 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, menstruation, pus, sebum, vomit, vaginal secretions, mucous secretion, fecal water, pancreatic juice, sinus cavity lavage fluids, bronchopulmonary aspirates, blastocillic cavity fluid, and umbilical cord blood).A sample may also 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, lymphatic fluid, outer sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs. An example also refers to a medium, such as a broth or nutrient gel, which may contain cellular components, such as proteins or nucleic acid molecules.

[0093] As used herein, the term “reference level” refers to a value from a “reference sample” or a “control sample” for determining the effect induced by the methods described herein. A reference level may be a metric or measurement determined before the administration or implementation of the method (e.g., one or more codon optimization methods or one or more treatment methods described herein). A reference level may be a metric or measure determined in a reference sample in which the methods described herein have not been administered (e.g., a negative control sample; for example, a healthy subject control or a subject with a disease or condition). A reference level may be a metric or measure determined in a Petition 870250098454, dated 10 / 28 / 2025, page 44 / 170 37 / 141 reference sample that exhibits a known or expected effect to assess the effectiveness of the methods described herein (e.g., effects produced by routine protein production methods or effects produced by known treatment methods). In some embodiments, a reference level may be a predetermined value or a value. As the skilled practitioner will be able to perceive, the reference level is predetermined and defined to meet requirements in terms of, for example, specificity and / or sensitivity. It may be, for example, that the sensitivity or specificity of the assay, respectively, have to be defined within 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 the subject belongs.In certain modalities, the reference level may be defined as any percentage between, for example, 25% and 75% of the overall distribution of values ​​in an investigated disease entity. In other modalities, the reference level may be defined as, for example, the median, tertiles, quartiles, or quintiles, as determined from the overall distribution of values ​​in an investigated disease entity or in a given population. In one modality, the reference level is defined as the median value determined from the overall distribution of values ​​in an investigated disease entity. In some modalities, the reference level may depend on the patient's sex; for example, men may have a different reference level than women.

[0094] As used herein, the terms “effective amount”, “therapeutically effective amount” and “sufficient amount” of a composition described herein refer to an amount sufficient for, when administered to the subject (e.g., a mammal; e.g., a subject Petition 870250098454, dated 10 / 28 / 2025, page 45 / 170 38 / 141 human), produce beneficial or desired results, including clinical results, and as such, an “effective amount” or synonym thereof depends on 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 on a number of factors, such as the therapeutic agent in question (e.g., a polynucleotide or a vector; e.g., an AAV comprising a transgene as described herein or a plurality thereof, an inhibitory RNA 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 the host to be treated, and the like, but may nevertheless be routinely determined by a specialist in the field.Furthermore, as used herein, a therapeutically effective amount of a composition of the present disclosure is an amount that results in a beneficial or desired outcome in a subject compared to a reference (e.g., the subject before treatment, a healthy control, or an untreated subject). As defined herein, a therapeutically effective amount of a composition of the present disclosure can be readily determined by a practitioner of ordinary skill by routine methods known in the art.

[0095] As used herein, the term “therapeutic level,” in reference to protein expression, refers to a level of protein expression that is within an acceptable range to confer a desired effect of a treatment. A therapeutic level may refer to a physiologically acceptable level of expression based on a healthy control subject or to a median level of expression based on a plurality of healthy control subjects. A therapeutic level may refer to a level of expression that falls within a range of acceptable expression levels based on subject characteristics such as age, sex, weight, height, ethnicity, lifestyle, and / or one or more comorbidities. A therapeutic level may be Petition 870250098454, dated 10 / 28 / 2025, p. 46 / 170 39 / 141 refer to an expression level that exceeds physiological values, such that the therapeutic expression level is approximately 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, or approximately 500% higher, compared to an expression level with based on a healthy control subject or a median level of expression based on a plurality of healthy control individuals.

[0096] As used herein, the term “subtherapeutic level,” in reference to protein expression, refers to a level of protein expression that is below a therapeutic level. A subtherapeutic level of protein expression may refer to an expression level below a physiologically acceptable expression level based on a healthy control subject or to a median expression level based on a plurality of healthy control subjects. A subtherapeutic level may refer to an expression level that falls within a range of acceptable expression levels based on subject characteristics such as, for example, age, sex, weight, height, ethnicity, lifestyle, and / or one or more comorbidities.A subtherapeutic level can refer to an expression level that is below a physiological value, such that the subtherapeutic expression level is approximately 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 subtherapeutic level can refer to an expression level that is below a therapeutic level, such that the subtherapeutic expression level is approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% lower than a therapeutic level.

[0097] As used herein, “administration” refers to the dispensing, distribution, or application of a composition of the disclosure to a Petition 870250098454, dated 10 / 28 / 2025, p. 47 / 170 40 / 141 subject by any suitable route for delivery of the composition (e.g., a polynucleotide or a vector; for example, an AAV comprising a transgene as described herein or a plurality thereof, an inhibitory RNA or a plurality thereof), to the desired site in the subject. Exemplary routes of administration include intramuscular, subcutaneous, intradermal, intravenous, intraperitoneal, intra-arterial, intraventricular, intranasal, intraorbital, intracranial or intraosseous administration.

[0098] 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 such an interval that there may be an overlap of an effect of each agent on the subject who needs it. In some modalities, the agents are administered at intervals of about 1 or more weeks, 1 or more days, 1 or more hours, or 1 or more minutes apart. In some modalities, the administrations of the agents are spaced sufficiently close to each other so that a combinatorial (e.g., a synergistic effect) is achieved.

[0099] As used in this document, the term “pharmaceutically acceptable” refers to compounds, materials, compositions and / or dosage forms that are, within the scope of good medical sense, suitable for use in contact with the tissues of a subject, such as a mammal (e.g., a human), without excessive toxicity, irritation, allergic response or other problem or complication, consistent with a reasonable benefit / risk ratio.

[0100] As used herein, the term “pharmaceutically acceptable excipient” refers to any ingredient other than active agents (e.g., as described herein) present in pharmaceutical compositions that has the properties of being substantially non-toxic and non-inflammatory in individuals.

[0101] As used in this document, the term composition Petition 870250098454, dated 10 / 28 / 2025, page 48 / 170 41 / 141 pharmaceutical 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, in order to treat, reduce the likelihood of or otherwise control a particular disease or condition affecting or likely to affect the subject. DETAILED DESCRIPTION

[0102] Compositions and methods for using heterologous gene expression technology are described herein. For example, the compositions and methods described herein are directed to expressing a polynucleotide comprising a transgene of interest in a host and subsequently reducing expression as needed by administering one or more inhibitory nucleic acid molecules.

[0103] Advantageously, the compositions and methods described herein allow for the temporal modulation of transgene expression. These compositions and methods of use can be applied to adjustable expression of a protein, such as a therapeutic protein, in the context of gene or cell therapies. I. Distribution of a Transgene for Heterologous Expression

[0104] Below are presented 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 a fragment thereof) and vectors or compositions and methods suitable for delivering said nucleic acid molecule to a host cell. All such compositions and pharmaceutical compositions and methods described are useful for treatment methods described herein. A. Transgene for Heterologous Expression

[0105] A transgene for heterologous gene expression can be Petition 870250098454, dated 10 / 28 / 2025, page 49 / 170 42 / 141 delivered to a host cell (e.g., a host cell in a subject who has or is at risk of having a disease or condition; e.g., 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 soluble protein, a transmembrane protein, a membrane-associated protein, an intracellular protein, a secreted protein, or a fragment thereof.

[0106] A transgene for heterologous expression can be administered to a host cell (e.g., a host cell in a subject who has or is at risk of having a disease or condition; e.g., 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 condition, such as a gene or a disease or condition presented in Table 1.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... Petition 870250098454, dated 10 / 28 / 2025, page 50 / 170 43 / 141 a fragment of it 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 in a subset of tissues or cell types contained therein.

[0107] In some embodiments, a transgene encodes a polypeptide, a protein, or a fragment thereof that shares 100% identity with 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 with 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 designed 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 an Fc fusion protein or an albumin fusion protein, which may further enhance protein stability, protein biodistribution, and / or protein half-life.

[0108] A polynucleotide sequence of a transgene for heterologous expression may be modified to include a binding site or a complementarity region for hybridization of one or more molecules. Petition 870250098454, dated 10 / 28 / 2025, page 51 / 170 44 / 141 of inhibitory RNA molecules. In preferred embodiments, the polynucleotide sequence of a transgene that includes a binding site or a complementarity region for hybridization of one or more inhibitory RNA molecules is present in the transgene mRNA transcript. In some embodiments, a region of a transgene is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% complementary to one or more inhibitory RNA molecules. In some embodiments, a region of a transgene contains a polynucleotide sequence complementary to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 contiguous sequences within a region of an inhibitory RNA molecule.

[0109] In some embodiments, the binding site or complementarity region for an inhibitory RNA molecule does not share sequence identity with an endogenous polynucleotide sequence of a host cell (e.g., a region of a host genome, a native nucleic acid; e.g., a native mRNA molecule). In some embodiments, the binding site or complementarity region for an inhibitory molecule is in an intron or a region therein of the transgene (e.g., the mRNA transcript of the transgene). In some embodiments, the binding site or complementarity region for an inhibitory RNA molecule is in an exon or a region therein of the transgene (e.g., the mRNA transcript of the transgene). In some embodiments, the binding site or complementarity region for an inhibitory molecule is in an exon or a region therein of the transgene (e.g., the mRNA transcript of the transgene).In some modalities, the linking site or region of complementarity is used. Petition 870250098454, dated 10 / 28 / 2025, p. 52 / 170 45 / 141 An inhibitory RNA molecule is located in an untranslated region (UTR) or a region within it of the transgene, such as the 5' UTR or the 3' UTR of the transgene (e.g., the transgene mRNA transcript). In some embodiments, the binding site or complementarity region for an inhibitory RNA molecule is approximately 10 bps, approximately 20 bps, approximately 30 bps, approximately 40 bps, approximately 50 bps, approximately 60 bps, approximately 70 bps, approximately 80 bps, approximately 90 bps, approximately 100 bps, approximately 110 bps, approximately 120 bps, approximately 130 bps, approximately 140 bps, or approximately 150 bps upstream of the transgene start codon (e.g., the transgene mRNA transcript).In some embodiments, the binding site or complementarity region for an inhibitory RNA molecule is approximately 10 bps, approximately 20 bps, approximately 30 bps, approximately 40 bps, approximately 50 bps, approximately 60 bps, approximately 70 bps, approximately 80 bps, approximately 90 bps, approximately 100 bps, approximately 110 bps, approximately 120 bps, approximately 130 bps, approximately 140 bps, or approximately 150 bps downstream of the stop codon of the transgene (e.g., the transgene mRNA transcript). B. Viral Genomes for Distributing a Transgene

[0110] Viral genomes provide a rich source of vectors that can be used for the efficient delivery of a transgene of interest into a host cell (e.g., a host cell in a subject who has or is at risk of having a disease or condition; e.g., 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 can be used in conjunction with the compositions and methods described herein are adeno-associated viruses (AAVs), retroviruses, adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35). Petition 870250098454, dated 10 / 28 / 2025, page 53 / 170 46 / 141 and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus) and poxviruses (e.g., vaccinia, modified Ankara vaccinia (MVA), avian pox and canary pox). Other viruses that may be used in conjunction with the compositions and methods described herein include Norwalk virus, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses and hepatitis viruses, for example. Examples of retroviruses include: avian leukosis-sarcoma, mammalian type C viruses, type B viruses, type D viruses, HTLV-BLV group, lentiviruses, foam viruses (Coffin, JM).(Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, BN Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). Other examples include murine leukemia virus, murine sarcoma virus, mouse mammalian tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, endogenous baboon viruses, gibbon primate 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 U.S. Patent No. 5,801,030, disclosure of which is incorporated herein by reference with respect to viral vectors for use in gene therapy. C. AAV Vectors for Delivering a Transgene of Interest

[0111] In some embodiments, a transgene of interest is incorporated into recombinant AAV (rAAV) vectors to facilitate its introduction into a cell. Useful rAAV vectors in conjunction with Petition 870250098454, dated 10 / 28 / 2025, page 54 / 170 47 / 141 compositions and methods described herein include recombinant nucleic acid constructs containing (1) a polynucleotide, such as a transgene encoding a protein or fragment thereof, and (2) one or more nucleic acids that facilitate the expression of the polynucleotide. Viral nucleic acids may include those cis-acting elements of rAAV for replication and packaging (e.g., functional ITRs) of DNA into a virion. Such rAAV vectors may also contain marker or reporter genes. Useful rAAV vectors include those that have one or more naturally occurring AAV genes deleted in whole or in part, but retain functional flanking ITR sequences. 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 Tal et al. (J. Biomed. Sci. 7:279-291, 2000), and Monahan and Samulski (Gene Delivery.7:24-30, 2000), the disclosures of each of them are incorporated here by reference, as they pertain to AAV vectors for gene administration.

[0112] The nucleic acids and vectors described in this document can be incorporated into an rAAV virion in order to facilitate the introduction of the nucleic acid or vector into a cell. The AAV capsid proteins comprise the non-nucleic acid outer 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 necessary for virion assembly. The rAAV virion construct has been described, for example, in U.S. Patents 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 (2002) and Bowles et al., J. Virol. 77:423-432 (2003), whose publications are incorporated herein by reference with regard to AAV vectors for gene delivery.

[0113] The rAAV virions useful in conjunction with the compositions and methods described herein include those derived from a variety of Petition 870250098454, dated 10 / 28 / 2025, p. 55 / 170 48 / 141 AAV serotypes, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 and AAV13, among others. the 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: 66156624, 2001), and Auricchio et al. (Hum. Molec. Genet. 10: 3075-3081, 2001), whose publications are incorporated herein by reference with regard to AAV vectors for gene delivery.

[0114] The nucleic acids and vectors described herein may also comprise a promoter sequence to initiate the 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, for example, a tetracycline-inducible promoter that induces gene expression in the presence of tetracycline or doxycycline. In some embodiments, the promoter is a naturally occurring promoter, so that the promoter is found in an organism (e.g., a target organism intended to receive the delivery or administration of one or more polynucleotides described herein). In some embodiments, the promoter occurs naturally, so that it is native to a gene comprising the transgene of interest. In some embodiments, the promoter is a synthetic promoter.Additional exemplary promoters that are useful for transgene expression include, but are not limited to, a respiratory syncytial virus (RSV) promoter, a cytomegalovirus (CMV) promoter, an elongation factor 1a (EF1a) 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, and a chicken elemental β-actin promoter. Petition 870250098454, dated 10 / 28 / 2025, page 56 / 170 49 / 141 CMV enhancers (CB7), a tetracycline-controlled transactivator protein (tTA) promoter, an upstream activation sequence (UAS) promoter, a homeobox 9 protein (HB9) promoter, a CD68 molecule promoter (CD68), a platelet-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.

[0115] Also useful in conjunction with the compositions and methods described in this document 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 different from the serotype provided (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13, 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 of a different AAV serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, or AAVrh74).Techniques involving the construct 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).

[0116] In some embodiments, the AAV comprises a capsid disclosed, for example, in WO 2017 / 218842, which disclosure is incorporated herein by reference. In some embodiments, the AAV comprises a capsid protein disclosed in Lin et al. (Mol Brain. 13:138, 2020), which disclosure is Petition 870250098454, dated 10 / 28 / 2025, page 57 / 170 50 / 141 incorporated herein by reference. In some embodiments, the AAV comprises an AAV2-retro or AAV9-retro capsid protein. In some embodiments, the AAV comprises a capsid protein that is conjugated to a linker or an aptamer.

[0117] AAV virions that have mutations within the virion capsid can be used to infect particular cell types more effectively than non-mutated capsid virions. For example, suitable AAV mutants may have ligand insertion mutations to facilitate AAV targeting to specific cell types. The construct 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 the methods of the invention include those capsid hybrids that are generated by molecular reproduction of viruses as well as by exon shuffling. See, for example, Soong et al. (Nat. Genet. 25:436439, 2000) and Kolman and Stemmer (Nat. Biotechnol. 19:423-428, 2001). D. Additional Methods for Delivering a Transgene to a Host Cell

[0118] Techniques that can be used to introduce a transgene, as described herein, into a host cell (e.g., a host cell in an individual with or at risk of developing a disease or condition; e.g., a human individual) are known in the art. For example, electroporation can be used to permeabilize mammalian cells (e.g., human cells) by applying an electrostatic potential to the cell of interest. Mammalian cells, such as human cells, subjected to an external electric field in this way are subsequently predisposed to the uptake of exogenous nucleic acids. Electroporation of mammalian cells is described in detail, for example, in Petition 870250098454, dated 10 / 28 / 2025, page 58 / 170 51 / 141 Chu et al. (Nucleic Acids Res. 15: 1311, 1987), whose disclosure is incorporated herein by reference. A similar technique, NUCLEOFECTION™, uses an applied electric field to stimulate the uptake of exogenous polynucleotides into the nucleus of a eukaryotic cell. NUCLEOFECTION™ and useful protocols for performing this technique are described in detail, for example, in Distler et al. (Exp. Dermatol. 14:315, 2005), as well as in US 2010 / 0317114, whose disclosures are incorporated herein by reference.

[0119] Additional useful techniques for target cell transfection include the compression poration methodology. This technique induces rapid mechanical deformation of cells in order to stimulate the uptake of exogenous DNA through membrane pores that form in response to applied stress. This technology is advantageous insofar as a vector is not required for the delivery of nucleic acids into a cell, such as a human target cell. Compression poration is described in detail, for example, in Sharei et al. (J. Vis. Exp. 81:e50980, 2013), the publication of which is incorporated herein by reference.

[0120] Lipofection represents another useful technique for transfecting target cells. This method involves loading nucleic acids into a liposome, which often features cationic functional groups such as quaternary or protonated amines, toward the exterior of the liposome. This promotes electrostatic interactions between the liposome and a cell due to the anionic nature of the cell membrane, which ultimately leads to the uptake of 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 U.S. Patent No. 7,442,386, disclosure of which is incorporated herein by reference. Similar techniques that exploit ionic interactions with the cell membrane to induce the uptake of foreign nucleic acids include contacting a cell with a Petition 870250098454, dated 10 / 28 / 2025, page 59 / 170 52 / 141 Cationic polymer-nucleic acid complex. Exemplary cationic molecules that associate with polynucleotides in order to confer a positive charge favorable to interaction with the cell membrane are activated dendrimers (described, for example, in Dennig (Topics in Current Chemistry 228:227, 2003), whose publication is incorporated here by reference) and diethylaminoethyl (DEAE)dextran, whose use as a transfection agent is described in detail, for example, in Gulick et al. (Curr. Protoc. in Mol. Biol. 40:I:9.2:9.2.1, 1997), whose publication is incorporated here by reference. Magnetic beads are another tool that can be used to transfect target cells gently and efficiently, as this methodology uses an applied magnetic field to direct the uptake of nucleic acids. This technology is described in detail, for example, in US 2010 / 0227406, the disclosure of which is incorporated herein by reference.

[0121] 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 specific wavelength to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. This technique is described in detail, for example, in Rhodes et al. (Methods in Cell Biology 82:309, 2007), the publication of which is incorporated herein by reference.

[0122] Microvesicles represent another potential vehicle that can be used to modify the genome of a target cell according to the methods described in this document. For example, microvesicles that have been induced by co-overexpression of the glycoprotein VSV-G with, for example, a genome-modifying protein, such as a nuclease, can be used to efficiently deliver proteins into a cell that subsequently catalyzes site-specific cleavage of an endogenous polynucleotide sequence in order to prepare the cell's genome for the Petition 870250098454, dated 10 / 28 / 2025, pp. 60 / 170 53 / 141 covalent incorporation of a polynucleotide of interest, such as a gene or regulatory sequence. The use of such vesicles, also called geicles, for the genetic modification of eukaryotic cells is described in detail, for example, in Quinn et al., Mol. Ther. 23: Supplement 1, Abstract No. 122, 2015). E. Genetic Engineering Methods of a Host Cell

[0123] In addition to the methods described herein, a host cell may be modified to comprise a transgene by means of 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), meganuclease, zinc finger nuclease, a CRISPR / Cas9-based approach, homologous recombination, primary editing, transposon-mediated delivery, in which an exogenous polynucleotide (e.g., a polynucleotide comprising a transgene encoding a protein of interest; e.g., a polynucleotide comprising a transgene and a complementarity region to one or more inhibitory nucleic acids) may be integrated into the genome of a host cell. i. CRISPR

[0124] A host cell can be modified to comprise a polynucleotide that includes a transgene and / or a region of complementarity to one or more inhibitory nucleic acids using Clustered Regularly Interleaved Short Palindromic Repeats (CRISPR) technology. “CRISPR” is a programmable technology that targets specific stretches of genetic code to edit DNA at precise locations. CRISPR technology may include CRISPR-Cas9. Cas9 (or CRISPR-associated protein 9) is a nuclease that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary. Petition 870250098454, dated 10 / 28 / 2025, page 61 / 170 54 / 141 to the CRISPR sequence, allowing 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's genome a transgene that codes for a protein of interest and a region that has complementarity with one or more inhibitory nucleic acids.

[0125] Exemplary CRISPR systems include those that utilize a Cas9 nuclease. The Cas9 nuclease, along with CRISPR sequences, forms the basis of a technology known as CRISPR-Cas9, which can be used to edit genes within organisms. CRISPR technology can include Class 1 CRISPR systems, including type I (Cas3), type III (Cas10), and type IV, and 12 subtypes. CRISPR technology can 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 similar tools. CRISPR technology can also be used as a diagnostic tool. For example, CRISPR-based diagnostics can be coupled with enzymatic processes, such as in vitro Transcription Sherlock-based Profiling (SPRINT). SPRINT can be used to detect a variety of substances, such as metabolites in individual samples or contaminants in environmental samples, with high throughput or with portable point-of-care devices.

[0126] Without being tied to the theory, the mechanism of action of certain CRISPR nucleases includes the step of forming an R loop, through the Petition 870250098454, dated 10 / 28 / 2025, page 62 / 170 55 / 141 which the CRISPR nuclease induces the unwinding of a double-stranded DNA target, thus separating the strands in the region bound by the CRISPR nuclease. The guide RNA spacer then hybridizes with the target strand in the protospacer sequence. This displaces a non-target strand that is complementary to the target strand, which forms the single-stranded region of the R loop.

[0127] 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 site and / or cuts the target strand at a second site. In some embodiments, a CRISPR nuclease may 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 killed, which cuts zero strands of a target nucleic acid.

[0128] In some embodiments, the CRISPR nuclease comprises any 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 of the amino acid sequences presented herein.

[0129] Examples of CRISPR nucleases include, without limitation, Cas9 (e.g., catalytically killed Cas9 (dCas9) and nickase Cas9 (nCas9)), Cas12a / Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i and Cas12j / CasΦ, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Petition 870250098454, dated 10 / 28 / 2025, p. 63 / 170 56 / 141 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, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Cas type II effector proteins, Cas type V effector proteins, Cas type VI effector proteins, CARF, DinG, homologs thereof, or modified or engineered versions thereof.

[0130] In some embodiments, the CRISPR system comprises a gene editor. In some embodiments, the gene editor comprises a CRISPR nuclease. The gene editor CRISPR nuclease can be any CRISPR nuclease described herein. In some embodiments, the gene editor CRISPR nuclease is an nCas. In some embodiments, the base editor CRISPR nuclease is a dCas. In some embodiments, the disclosure gene editor further comprises an NLS (nuclear localization signal) domain.

[0131] In some embodiments, the gene editor is a fusion protein, and the gene editor components are domains of the fusion protein, optionally connected by a linker. In some embodiments, the gene editor is a multiprotein complex, and the gene editor components are provided as individual polypeptides. In some embodiments, the gene editor is a multiprotein complex, and one or more gene editor components are provided endogenously by the cell.

[0132] In some embodiments, the CRISPR system comprises a master editor. In some embodiments, the master editor comprises a CRISPR nuclease and a reverse transcriptase (RT) polypeptide. In some embodiments, the gRNA for use with the master editor is a master-editing gRNA (PEgRNA). The gene editor's CRISPR nuclease may be any CRISPR nuclease described herein. In some embodiments, the gene editor's CRISPR nuclease is an nCas. In some embodiments, the nuclease Petition 870250098454, dated 10 / 28 / 2025, p. 64 / 170 57 / 141 The CRISPR base editor is a dCas. In some embodiments, the base editors further comprise a flap endonuclease polypeptide. In some embodiments, the flap endonuclease polypeptide of the base editor is a FEN1 domain. In some embodiments, the base editors further comprise an NLS sequence.

[0133] In some embodiments, the master editor is a fusion protein, and the master editor components are domains of the fusion protein, optionally connected by a linker. In some embodiments, the master editor is a multiprotein complex, and the master editor components are provided as individual polypeptides. In some embodiments, the master editor is a multiprotein complex, and one or more master editor components are provided endogenously by the cell. ii. Transposon System

[0134] A host cell can be modified to comprise a polynucleotide comprising a transgene and / or a region of complementarity to one or more inhibitory nucleic acids using a transposon system. Transposons or transposable elements include a (short) nucleic acid sequence with terminal repeat sequences upstream and downstream of it. Transposons are polynucleotides that encode transposase enzymes and contain a polynucleotide sequence (e.g., a polynucleotide sequence comprising a transgene and a region of complementarity to one or more inhibitory nucleic acids) flanked by excision sites at the 5' and 3' positions. After a transposon is delivered into a cell, expression of the transposase gene begins and results in active enzymes that cleave the transposon polynucleotide. This activity is mediated by site-specific recognition of transposon excision sites by the transposase.

[0135] In some modalities, these excision sites may Petition 870250098454, dated 10 / 28 / 2025, page 65 / 170 58 / 141 being terminal repeats or inverted terminal repeats. Once excised from the transposon, the polynucleotide (for example, that polynucleotide comprising a transgene and a region that has complementarity with one or more inhibitory nucleic acids) can be integrated into the genome of a host cell by transposase-catalyzed cleavage of similar excision sites that exist within the cell's nuclear genome. This allows the polynucleotide to be inserted into the cleaved nuclear DNA at the excision sites, and the subsequent linking of phosphodiester bonds that link the polynucleotide of interest to the host cell's genomic DNA completes the incorporation process.In some embodiments, the transposon may be a retrotransposon, such that the polynucleotide (e.g., a DNA sequence comprising a transgene and a region that has complementarity with one or more inhibitory nucleic acids) is first transcribed to an RNA product and then reverse transcribed to DNA before incorporation into the genome of the prokaryotic or eukaryotic cell. Exemplary transposon systems include the piggyBac transposon, the Sleeping Beauty transposon, the Frog Prince transposon, and the Tol2 transposon. II. Inhibitory Nucleic Acids

[0136] Below are presented inhibitory nucleic acid molecules, including small interfering RNAs, double-stranded RNAs, microRNAs, short hairpin RNAs, antisense oligonucleotides and gapmers, as well as exemplary modifications thereof that can be used to reduce the expression of a transgene or an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) delivered by one or more of the vectors (e.g., AAV vectors) described herein. All such inhibitory nucleic acid molecules, modifications thereof and pharmaceutical compositions thereof are useful for methods such as the methods of Petition 870250098454, dated 10 / 28 / 2025, pp. 66 / 170 59 / 141 treatment, described here. A. Small interfering RNAs

[0137] A small interfering RNA (siRNA) molecule is a single-stranded (ss) or double-stranded (ds) nucleic acid molecule made of DNA, RNA, or DNA-RNA nucleosides (e.g., a chimeric) that is complementary to an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) or a region therein, and prevents the translation of the mRNA into a protein. When an siRNA molecule enters a cell, it is incorporated into an RNA-induced silencing complex (RISC). After hybridization of an antisense strand of the siRNA molecule with a region on an mRNA molecule after transgene transcription, the RISC complex will cleave the target mRNA, thus inactivating the target mRNA, resulting in reduced levels of target mRNA and protein.In some cases, an siRNA inhibits the translation of mRNA into a protein through translation repression or deadenylation-dependent decay mechanisms.

[0138] In some embodiments, an siRNA molecule may include a nucleotide sequence of about 10 to about 30 nucleotides in length (e.g., 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 or 31 nucleotides in length).

[0139] In some embodiments, a disclosure siRNA molecule may include a nucleotide sequence of 10 to 30 nucleotides in length (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length).

[0140] It is within the scope of disclosure that any Petition 870250098454, dated 10 / 28 / 2025, pp. 67 / 170 60 / 141 length, known and previously unknown in the art, can be implemented for the present invention.

[0141] In some embodiments, an siRNA molecule contains an antisense strand. In some embodiments, the length of the antisense strand is between 10 and 30 nucleotides (e.g., 10 nucleotides, 11 nucleotides, nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 21 nucleotides, 25 nucleotides, 18 nucleotides, 22 nucleotides, 26 nucleotides, 19 nucleotides, 23 nucleotides, 27 nucleotides, 20 nucleotides, 24 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), 15 and 25 nucleotides (e.g., 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides or 30 nucleotides), or 18 and 23 nucleotides (for example, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides or 23 nucleotides).In some embodiments, the antisense strand is 20 nucleotides long. In some embodiments, the antisense strand is 21 nucleotides long. In some embodiments, the antisense strand is 22 nucleotides long. In some embodiments, the antisense strand is 23 nucleotides long. In some embodiments, the antisense strand is 24 nucleotides long. In some embodiments, the antisense strand is 25 nucleotides long. In some embodiments, the antisense strand is 26 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long.

[0142] In some embodiments, an siRNA molecule contains a sense strand. In some embodiments, the sense strand is between 10 and 30 nucleotides (e.g., 10 nucleotides, 11 nucleotides, 12 nucleotides, Petition 870250098454, dated 10 / 28 / 2025, page 68 / 170 61 / 141 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 22 nucleotides, 23 nucleotides, 26 nucleotides, 27 nucleotides, or 30 nucleotides), or nucleotides, 15 nucleotides, 16 nucleotides, 19 nucleotides, 20 nucleotides, 20 nucleotides, 21 nucleotides, 24 nucleotides, 25 nucleotides, 28 nucleotides, 29 and 23 nucleotides (for example, 14 nucleotides, 17 nucleotides, 18 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides). In some embodiments, the sense strand is 15 nucleotides. In some embodiments, the sense strand has 16 nucleotides. In some embodiments, the sense strand is 17 nucleotides long. In some embodiments, the sense strand is 18 nucleotides long. In some embodiments, the sense strand is 19 nucleotides long. In some embodiments, the sense strand is 20 nucleotides long. In some embodiments, the sense strand is 21 nucleotides long. In some embodiments, the sense strand is 22 nucleotides long.In some embodiments, the sense strand has 23 nucleotides. In some embodiments, the sense strand has 24 nucleotides. In some embodiments, the sense strand has 25 nucleotides. In some embodiments, the sense strand has 26 nucleotides. In some embodiments, the sense strand has 27 nucleotides. In some embodiments, the sense strand has 28 nucleotides. In some embodiments, the sense strand has 29 nucleotides. In some embodiments, the sense strand has 30 nucleotides.

[0143] In some embodiments, the sense and antisense strands of an siRNA molecule are completely complementary to each other (e.g., 100% complementary). In some embodiments, the sense and antisense strands of a disclosure siRNA molecule are completely complementary to the extent that their lengths overlap. Depending on the sequence of the sense and antisense strands, the complementarity need not be complete or perfect, meaning that the Petition 870250098454, dated 10 / 28 / 2025, pp. 69 / 170 62 / 141 sense and antisense strands do not hybridize at all base pairs due to mismatches. One or more (e.g., 1, 2, 3, 4, or 5) mismatches may be present in the ds siRNA molecule without affecting the siRNA molecule's ability to reduce the expression of an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) and ultimately the expression of a protein.

[0144] In some embodiments, an siRNA molecule contains a sequence complementary to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 nucleotides contiguous to a segment of equal length within a region of an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) with which the siRNA molecule can hybridize with the mRNA transcript or a region contained therein.

[0145] The nucleotide sequence of an siRNA molecule may contain sufficient complementarity to a region of an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., a transcript encoding a protein of interest) such that the siRNA molecule can hybridize with a region unique to the transcript and not hybridize with a native or endogenous region of a host cell (e.g., a host genome; e.g., the genome of a subject). In some embodiments, the siRNA may hybridize with one or more regions unique to the transcript. In some embodiments, the siRNA molecule is at least 70%, at least 75%, at least 80%, at least 85%, by Petition 870250098454, dated 10 / 28 / 2025, pp. 70 / 170 63 / 141 less than 90%, at least 95%, at least 98%, or at least 99% complementary to an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) or to a region (e.g., a segment of equal length). In some embodiments, the siRNA molecule is 100% complementary to an mRNA transcript (e.g., a transcript transcribed from the transgene of interest; e.g., a transcript encoding a protein of interest) or to a region contained therein.

[0146] In some embodiments, the nucleotide sequence of an siRNA molecule may contain sufficient complementarity to an exon sequence within an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) or a region therein. In some embodiments, the nucleotide sequence of an siRNA molecule may contain sufficient complementarity to an intron sequence within a transcript encoding a protein of interest, or a portion thereof. In some embodiments, an siRNA molecule may contain sufficient complementarity to a pre-mRNA transcript or to an mRNA transcript of a protein of interest. In some embodiments, the nucleotide sequence of an siRNA molecule may contain sufficient complementarity to an untranslated region (UTR) of the mRNA transcript encoding a protein of interest.In some embodiments, the UTR of the mRNA transcript is the 5' UTR. In some embodiments, the UTR of the mRNA transcript is the 3' UTR. In some embodiments, an siRNA molecule may contain sufficient complementarity to a region upstream of the start codon of an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest), such that the siRNA molecule is complementary to a region that is upstream of the start codon of an mRNA transcript (e.g., an mRNA transcript encoding a protein of interest), such that the siRNA molecule is complementary to a region that is upstream of the start codon of an mRNA transcript (e.g., an mRNA transcript encoding a protein of interest). Petition 870250098454, dated 10 / 28 / 2025, pp. 71 / 170 64 / 141 approximately 10 bps, approximately 20 bps, approximately 30 bps, approximately 40 bps, approximately 50 bps, approximately 60 bps, approximately 70 bps, approximately 80 bps, approximately 90 bps, approximately 100 bps, approximately 110 bps, approximately 120 bps, approximately 130 bps, approximately 140 bps, or approximately 150 bps upstream of the transcript start codon.In some embodiments, an siRNA molecule may contain sufficient complementarity to a region downstream of the stop codon of an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest), such that the siRNA molecule is complementary to a region that is approximately 10 bps, approximately 20 bps, approximately 30 bps, approximately 40 bps, approximately 50 bps, approximately 60 bps, approximately 70 bps, approximately 80 bps, approximately 90 bps, approximately 100 bps, approximately 110 bps, approximately 120 bps, approximately 130 bps, approximately 140 bps, or approximately 150 bps downstream of the stop codon of the mRNA transcript.

[0147] In some embodiments, a siRNA molecule described herein may have 0-7 nucleotide 3' overhangs or 0-4 nucleotide 5' overhangs. In some embodiments, the siRNA molecule has a single uracil overhang at one or more 3' ends of the siRNA. In some embodiments, the siRNA molecule has a double uracil overhang at one or more 3' ends of the siRNA. In some embodiments, the siRNA molecule has a single uracil overhang at one or more 3' ends of the siRNA. In some embodiments, the siRNA molecule has a double thymine overhang at one or more 3' ends of the siRNA. In some embodiments, the siRNA molecule has a single cytosine and a single thymine (e.g., CT) overhang at one or more 3' ends of the siRNA.

[0148] For any of the methods described here, different siRNA molecules (e.g., two or more, three or more, four or more, or five or more different siRNA molecules) can be combined to reduce the expression of a transgene of interest (e.g., a transgene Petition 870250098454, dated 10 / 28 / 2025, p. 72 / 170 65 / 141 therapeutic that encodes a protein of interest; for example, a transgene embedded in an AAV vector described herein). A combination of two or more siRNA molecules, such as two different siRNA molecules, three different siRNA molecules, four different siRNA molecules, five or more different siRNA molecules, may have overlapping regions of complementarity to an mRNA transcript (for example, an mRNA transcript transcribed from the transgene of interest; for example, an mRNA transcript encoding a protein of interest) and may be used in a disclosure method to inhibit said transcript.Alternatively, two different siRNA molecules, three different siRNA molecules, four different siRNA molecules, five different siRNA molecules, or more, may not have any overlapping regions of complementarity to an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) and are instead complementary to distinct regions of a transcript and may be used in a disclosure method to inhibit the mRNA transcript. B. Double-stranded RNAs

[0149] A disclosure double-stranded RNA (dsRNA) molecule is a double-stranded nucleic acid molecule made of DNA, RNA, or DNA-RNA nucleosides (e.g., a chimeric) that is complementary to an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) and prevents translation of the mRNA into a protein or polypeptide. Typically, a dsRNA molecule is larger than an siRNA molecule and is processed within a cell to form an siRNA molecule. The antisense strand of the siRNA molecule is then incorporated into the RISC, which, after hybridization of the siRNA with a target mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) Petition 870250098454, dated 10 / 28 / 2025, page 73 / 170 66 / 141 For example, an mRNA transcript encoding a protein of interest will cleave the target mRNA transcript, thus inactivating the target mRNA transcript and resulting in reduced levels of expression of both the mRNA transcript and the protein (e.g., the protein of interest). In some cases, a dsDNA can prevent the translation of mRNA into a protein through translation repression mechanisms.

[0150] In some embodiments, a disclosure dsRNA molecule may include a sense strand and an antisense strand, each containing a nucleotide sequence of about 25 to about 5,000 nucleotides in length, or more.

[0151] It is within the scope of disclosure that any length, known and previously unknown in the art, can be implemented for the present invention.

[0152] In some embodiments, a disclosure dsRNA molecule contains a sequence complementary to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 contiguous nucleotides within a region of an mRNA transcript (for example, an mRNA transcript transcribed from the transgene of interest; for example, an mRNA transcript encoding a protein of interest) with which the dsRNA molecule can hybridize.

[0153] In some embodiments, the sense and antisense strands of a dsRNA molecule are completely complementary to each other. In some embodiments, the sense and antisense strands of a disclosure dsRNA molecule are completely complementary to the extent that their lengths overlap. Depending on the sequence of the sense and antisense strands Petition 870250098454, dated 10 / 28 / 2025, pp. 74 / 170 67 / 141 antisense, complementarity does not need to be complete or perfect, which means that the sense and antisense ribbons do not hybridize on all base pairs due to incompatibilities. One or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) mismatches may be present in the dsRNA molecule without affecting the dsRNA molecule's ability to reduce the expression of an mRNA transcript (e.g., an mRNA transcript transcribed from transgene of interest; for example, an mRNA transcript that codes for a protein of interest) and, finally, the expression of a protein.

[0154] The nucleotide sequence of a disclosure dsRNA molecule may contain sufficient complementarity to a portion of an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) such that the dsRNA molecule can hybridize with the mRNA transcript or a region contained therein. The nucleotide sequence of a dsRNA molecule may contain sufficient complementarity to a region of an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., a transcript encoding a protein of interest) such that the dsRNA molecule can hybridize with a region unique to the transcript and not hybridize with a native or endogenous region of a host cell (e.g., a host genome; e.g., the genome of a subject).

[0155] In some embodiments, the dsRNA molecule is at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least Petition 870250098454, dated 10 / 28 / 2025, pp. 75 / 170 68 / 141 99% complementary to an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) or a portion thereof. In some embodiments, the dsRNA molecule is 100% complementary to an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) or a region contained therein.

[0156] In some embodiments, the nucleotide sequence of a dsRNA molecule may contain sufficient complementarity to an exon sequence within an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) or a region therein. In some embodiments, the nucleotide sequence of a dsRNA molecule may contain sufficient complementarity to an intron sequence within an mRNA transcript encoding a protein of interest, or a portion thereof. In some embodiments, a dsRNA molecule may contain sufficient complementarity to a pre-mRNA transcript or an mRNA transcript encoding a protein of interest. In some embodiments, the nucleotide sequence of a dsRNA molecule may contain sufficient complementarity to an untranslated region (UTR) of the mRNA transcript encoding a protein of interest.In some embodiments, the UTR of the mRNA transcript is the 5' UTR. In some embodiments, the UTR of the mRNA transcript is the 3' UTR. In some embodiments, a dsRNA molecule may contain sufficient complementarity to a region upstream of the start codon of an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest), such that the siRNA molecule is complementary to a region that is approximately 10 bps, approximately 20 bps, approximately. Petition 870250098454, dated 10 / 28 / 2025, pp. 76 / 170 69 / 141 of 30 bps, approximately 40 bps, approximately 50 bps, approximately 60 bps, approximately 70 bps, approximately 80 bps, approximately 90 bps, approximately 100 bps, approximately 110 bps, approximately 120 bps, approximately 130 bps, approximately 140 bps, or approximately 150 bps upstream of the transcript start codon. In some embodiments, a dsRNA molecule may contain sufficient complementarity to a region downstream of the stop codon of an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest), such that the dsRNA molecule is complementary to a region that is approximately 10 bps, approximately 20 bps, approximately 30 bps, approximately 40 bps, approximately 50 bps, approximately 60 bps, approximately 70 bps, approximately 80 bps, approximately 90 bps, approximately 100 bps, approximately 110 bps, approximately 120 bps, approximately 130 bps, approximately 140 bps, or approximately 150 bps downstream of the stop codon of the mRNA transcript.

[0157] For any of the methods described herein, different dsRNA molecules (e.g., two or more, three or more, four or more, or five or more different dsRNA molecules) can be combined to reduce the expression of a transgene of interest (e.g., a therapeutic transgene encoding a protein of interest; e.g., a transgene incorporated into an AAV vector described herein). A combination of two or more dsRNA molecules, such as two different siRNA molecules, three different dsRNA molecules, four different dsRNA molecules, five or more different dsRNA molecules, may have overlapping regions of complementarity to an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) and may be used in a disclosure method to inhibit said transcript.Alternatively, two different siRNA molecules, three different siRNA molecules, four different dsRNA molecules, five different dsRNA molecules, or more, may not. Petition 870250098454, dated 10 / 28 / 2025, page 77 / 170 70 / 141 have no overlapping region of complementarity to an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) and are instead complementary to distinct regions of an mRNA transcript and can be used in a disclosure method to inhibit the mRNA transcript. C. MicroRNAs

[0158] A disclosure microRNA (miRNA) molecule is a short nucleic acid molecule made of DNA, RNA, or DNA-RNA nucleosides (e.g., a chimeric) that is complementary to an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) or a region therein and prevents translation of the mRNA into a protein or polypeptide. Once a miRNA molecule enters a cell, it is incorporated into the RISC, which, after hybridization of the miRNA with a target mRNA (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest), will cleave the target mRNA, thus inactivating the target mRNA and resulting in reduced levels of expression of the mRNA transcript and the protein (e.g., the protein of interest).

[0159] In some embodiments, a disclosure miRNA molecule may include a nucleotide sequence of about 6 to about 30 nucleotides in length (e.g., 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, approximately 27, approximately 28, approximately 29, approximately 30 or 31 nucleotides in length). Petition 870250098454, dated 10 / 28 / 2025, pp. 78 / 170 71 / 141

[0160] In some embodiments, a miRNA may include a nucleotide sequence of 6 to 30 nucleotides in length (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length).

[0161] It is within the scope of disclosure that any length, known and previously unknown in the art, can be implemented for the present invention.

[0162] In some embodiments, a miRNA molecule contains a sequence complementary to at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 contiguous nucleotides within a region of an mRNA transcript (for example, an mRNA transcript transcribed from the transgene of interest and encoding the protein of interest). The nucleotide sequence of the miRNA molecule may contain sufficient complementarity to a region of an mRNA transcript (for example, an mRNA transcript transcribed from the transgene of interest; for example, an mRNA transcript encoding a protein of interest) so that the miRNA molecule can hybridize with the mRNA transcript.The nucleotide sequence of a miRNA molecule may contain sufficient complementarity to a region of an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., a transcript encoding a protein of interest) such that the miRNA molecule can hybridize with a region unique to the transcript and not hybridize with a native or endogenous region of a host cell (e.g., a host genome; e.g., the genome of a subject). In some embodiments, the siRNA can hybridize with... Petition 870250098454, dated 10 / 28 / 2025, pp. 79 / 170 72 / 141 one or more unique regions of the transcript. In some embodiments, the siRNA molecule is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% complementary to an mRNA transcript (e.g., an mRNA transcript from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) or to a region (e.g., a segment of equal length). In some embodiments, the siRNA molecule is 100% complementary to a transcript (e.g., a transcript from the transgene of interest; e.g., a transcript encoding a protein of interest) or to a region contained therein.

[0163] In some embodiments, the miRNA molecule is at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) or a region thereon. In some embodiments, the miRNA molecule is 100% complementary to an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) or a region contained therein.

[0164] In some embodiments, the nucleotide sequence of a miRNA molecule may contain sufficient complementarity to an exon sequence within an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) or a region therein. In some embodiments, the nucleotide sequence of a miRNA molecule Petition 870250098454, dated 10 / 28 / 2025, pp. 80 / 170 73 / 141 miRNA may contain sufficient complementarity to an intron sequence within an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) or a region therein. In some embodiments, a disclosure miRNA molecule may contain sufficient complementarity to a pre-mRNA transcript or to an mRNA transcript within an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) or a region therein.

[0165] For any of the methods described herein, different miRNA molecules (e.g., two or more, three or more, four or more, or five or more different miRNA molecules) can be combined to reduce the expression of a transgene of interest (e.g., a therapeutic transgene encoding a protein of interest; e.g., a transgene incorporated into an AAV vector described herein). A combination of two or more miRNA molecules, such as two different miRNA molecules, three different miRNA molecules, four different miRNA molecules, five or more different miRNA molecules, may have overlapping regions of complementarity to an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) and may be used in a disclosure method to inhibit said transcript.Alternatively, two different miRNA molecules, three different miRNA molecules, four different miRNA molecules, five different miRNA molecules, or more, may not have any overlapping regions of complementarity to an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) and are instead complementary to distinct regions of an mRNA transcript. Petition 870250098454, dated 10 / 28 / 2025, page 81 / 170 74 / 141 can be used in a disclosure method to inhibit mRNA transcript. D. Short Hairpin RNAs

[0166] A short hairpin RNA (shRNA) disclosure molecule is an ss or ds nucleic acid molecule made of DNA, RNA, or DNA and RNA nucleosides (e.g., a chimeric) that is complementary to an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) or a region therein, and prevents translation of the mRNA transcript into a protein or polypeptide. Once an shRNA molecule enters a cell, it is incorporated into a RISC, which, after hybridization of the shRNA with a target mRNA (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) or a region therein, the RISC complex will cleave the target mRNA, thus inactivating the target mRNA and resulting in reduced levels of target mRNA and protein.

[0167] In some embodiments, a disclosure shRNA molecule may include a nucleotide sequence of about 60 to about 100 nucleotides in length (e.g., 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105 or 110 nucleotides in length).

[0168] In some embodiments, a disclosure shRNA molecule may include a nucleotide sequence of 60 to 100 nucleotides in length (e.g., 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleotides in length).

[0169] In some embodiments, an shRNA molecule of Petition 870250098454, dated 10 / 28 / 2025, p. 82 / 170 75 / 141 disclosure may contain a variable hairpin loop structure and a stem sequence. In some embodiments, the stem sequence may be 10 to 50 nucleotides long (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long). In some embodiments, the hairpin size is between 4 and 50 nucleotides in length (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length), although the loop size may be larger without significantly affecting silencing activity.An shRNA molecule may contain mismatches, for example, GU mismatches between two strands of the shRNA stem, without diminishing the potency or ability to reduce the expression levels of an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest). In some embodiments, an shRNA molecule is designed to include one or more GU pairs in the hairpin stem to stabilize the hairpins during propagation, for example.

[0170] The nucleotide sequence of a disclosure shRNA molecule may contain sufficient complementarity with an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; for example, an mRNA transcript encoding a protein of interest) or a region therein such that the shRNA molecule can hybridize with the mRNA transcript. The nucleotide sequence of a shRNA molecule may contain sufficient complementarity to a region of an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; for example, a transcript encoding a protein of interest) such that the shRNA molecule can hybridize with a unique region of the Petition 870250098454, dated 10 / 28 / 2025, page 83 / 170 76 / 141 transcribed and not hybridizing with a native or endogenous region of a host cell (e.g., a host genome; e.g., the genome of a subject). In some embodiments, the shRNA molecule is 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%, or at least 99% complementary to an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) or a region therein. In some embodiments, the shRNA molecule is 100% complementary to an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) or a region therein.

[0171] In some embodiments, the nucleotide sequence of a disclosure shRNA molecule may contain sufficient complementarity to an exon sequence within an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; for example, an mRNA transcript encoding a protein of interest) or a region therein. In some embodiments, the nucleotide sequence of a disclosure shRNA molecule may contain sufficient complementarity to an intron sequence within an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; for example, an mRNA transcript encoding a protein of interest) or a region therein.In some embodiments, a disclosure shRNA molecule may contain sufficient complementarity to a pre-mRNA transcript or an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) or a region therein.

[0172] For any of the methods described here, different Petition 870250098454, dated 10 / 28 / 2025, p. 84 / 170 77 / 141 shRNA molecules (e.g., two or more, three or more, four or more, or five or more different shRNA molecules) can be combined to reduce the expression of a transgene of interest (e.g., a therapeutic transgene encoding a protein of interest; e.g., a transgene incorporated into an AAV vector described herein). A combination of two or more shRNA molecules, such as two different shRNA molecules, three different shRNA molecules, four different shRNA molecules, five or more different shRNA molecules, may have overlapping regions of complementarity to an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) and may be used in a disclosure method to inhibit said transcript.Alternatively, two different shRNA molecules, three different shRNA molecules, four different shRNA molecules, five different shRNA molecules, or more, may not have any overlapping regions of complementarity to an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) and are instead complementary to distinct regions of a transcript and may be used in a disclosure method to inhibit the mRNA transcript. E. Antisense Oligonucleotides

[0173] An antisense oligonucleotide (ASO) is a single-stranded nucleic acid molecule containing DNA nucleosides that are complementary to an mRNA transcript (e.g., an mRNA transcript from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) or a region therein, and prevents the translation of the mRNA into a protein or polypeptide. After hybridization of an ASO with a target mRNA (e.g., an mRNA transcript from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest), the Petition 870250098454, dated 10 / 28 / 2025, page 85 / 170 78 / 141 RNase H will degrade mRNA by hydrolysis, resulting in reduced levels of target mRNA and protein.

[0174] In some embodiments, an ASO disclosure may include a nucleotide sequence of about 12 to about 50 nucleotides in length (e.g., 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, approximately 46, approximately 47, approximately 48, approximately 49, approximately 50 or 51 nucleotides in length).

[0175] In some embodiments, an ASO of the disclosure may include a nucleotide sequence of 12 to 50 nucleotides in length (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length).

[0176] It is within the scope of disclosure that any length, known and previously unknown in the art, can be implemented for the present invention.

[0177] In some embodiments, an ASO of the disclosure contains a sequence complementary to at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, Petition 870250098454, dated 10 / 28 / 2025, page 86 / 170 79 / 141 at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50 nucleotides contiguous to a segment of equal length within a region of an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) with which ASO can hybridize with the mRNA transcript or a region contained therein.

[0178] The nucleotide sequence of an ASO may contain sufficient complementarity to a region of an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., a transcript encoding a protein of interest) such that the ASO can hybridize to a region unique to the transcript and not to a region native to a host genome (e.g., the genome of a subject). In some embodiments, the ASO may hybridize to one or more regions unique to the mRNA transcript.In some embodiments, the ASO is at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., a transcript encoding a protein of interest) or a region contained therein. In some embodiments, the ASO is 100% complementary to an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., a transcript encoding a protein of interest) or a region contained therein.

[0179] In some embodiments, the nucleotide sequence of an ASO of disclosure may contain sufficient complementarity to a Petition 870250098454, dated 10 / 28 / 2025, page 87 / 170 80 / 141 exon sequence within an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., a transcript encoding a protein of interest) or a region contained therein. In some embodiments, the nucleotide sequence of an ASO may contain sufficient complementarity to an intron sequence within an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., a transcript encoding a protein of interest) or a region contained therein. In some embodiments, a disclosure ASO may contain sufficient complementarity to a pre-mRNA transcript or an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., a transcript encoding a protein of interest) or a region contained therein.

[0180] For any of the methods described herein, different ASOs (e.g., two or more, three or more, four or more, or five or more different ASOs) can be combined to reduce the expression of a transgene of interest (e.g., a therapeutic transgene encoding a protein of interest; e.g., a transgene incorporated into an AAV vector described herein). A combination of two or more ASOs, such as two different ASOs, three different ASOs, four different ASOs, five or more different ASOs, may have overlapping regions of complementarity to an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) and may be used in a disclosure method to inhibit said transcript.Alternatively, two different ASOs, three different ASOs, four different ASOs, five or more different ASOs may not have any overlapping region of complementarity to an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) and are, instead, Petition 870250098454, dated 10 / 28 / 2025, page 88 / 170 81 / 141 are complementary to distinct regions of a transcript and can be used in a disclosure method to inhibit the mRNA transcript. F. Gapmers

[0181] A gapmer is a single-stranded nucleic acid molecule containing an inner region of DNA (i.e., a gap segment) flanked by one or two outer regions of RNA (i.e., wing segments). At a minimum, the gap segment contains a sequence complementary to an mRNA transcript (e.g., an mRNA transcript from the transgene of interest; e.g., a transcript encoding a protein of interest) or a region contained therein. Typically, the wing segments contain modified RNA; the modified RNA (and DNA) is described further below. Upon hybridization of a gapmer with a target mRNA (e.g., an mRNA transcript from the transgene of interest; e.g., a transcript encoding a protein of interest) or a region therein, RNase H will degrade the mRNA transcript by hydrolysis, resulting in reduced levels of target mRNA and protein.

[0182] In some embodiments, a disclosure gapmer may include a nucleotide sequence of about 10 to about 25 nucleotides in length (e.g., 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25 or 26 nucleotides in length).

[0183] In some embodiments, a gapmer may include a nucleotide sequence of 10 to 25 nucleotides in length (for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length).

[0184] It is within the scope of disclosure that any length, known and previously unknown in the art, may be Petition 870250098454, dated 10 / 28 / 2025, p. 89 / 170 82 / 141 implemented for the present invention.

[0185] In some embodiments, a gapmer of the disclosure contains at least one (e.g., 1 or 2) wing region. In some embodiments, the gapmer contains a wing region located 5' relative to the gap region. In some embodiments, the gapmer contains a wing region located 3' relative to the gap region. In some embodiments, the gapmer contains two wing regions, one located 5' from the gap region and the other located 3' from the gap region.

[0186] In some embodiments, a gapmer contains at least one (e.g., 1 or 2) wing region that is about 1 to about 7 nucleotides long (e.g., about 1-8, about 1-7, about 1-6, about 1-5, about 1-4, about 1-3, or about 1-2 nucleotides long). In some embodiments, the gapmer contains at least one (e.g., 1 or 2) wing region that is 1 nucleotide long. In some embodiments, the gapmer contains at least one (e.g., 1 or 2) wing region that is 2 nucleotides long. In some embodiments, the gapmer contains at least one (e.g., 1 or 2) wing region that is 3 nucleotides long. In some embodiments, the gapmer contains at least one (e.g., 1 or 2) wing region with a length of 4 nucleotides. In some embodiments, the gapmer contains at least one (e.g., 1 or 2) wing region with a length of 5 nucleotides.In some embodiments, the gapmer contains at least one (e.g., 1 or 2) wing region with a length of 6 nucleotides. In some embodiments, the gapmer contains at least one (e.g., 1 or 2) wing region with a length of 7 nucleotides. In some embodiments, the gapmer contains at least one (e.g., 1 or 2) wing region with a length of 8 nucleotides.

[0187] In some forms, a gapper of the disclosure contains a gap region. In some forms, the gapper contains a Petition 870250098454, dated 10 / 28 / 2025, pp. 90 / 170 83 / 141 gap region located 5' from the wing region. In some forms, the gapmer contains a gap region located 3' from the wing region. In some forms, the gapmer contains a gap region flanked by two wing regions.

[0188] In some embodiments, a gapper of the disclosure contains at least one gap region that is about 8 to about 24 nucleotides long (for example, about 8-24, about 8-23, about 8-22, about 8-21, about 8-20, about 8-19, about 8-18, about 8-17, about 8-16, about 8-15, about 8-14, about 8-13, about 8-12, about 8-10, or about 8-9 nucleotides long).

[0189] The nucleotide sequence of the gapmer may contain sufficient complementarity to a portion of an mRNA transcript (for example, an mRNA transcript transcribed from the transgene of interest; for example, a transcript encoding a protein of interest) or a region contained therein, such that the gapmer can hybridize with the mRNA transcript or a region contained therein. In some embodiments, the gapmer is at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., a transcript encoding a protein of interest) or a region therein.In some embodiments, the gapmer is 100% complementary to an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., a transcript encoding a protein of interest) or a region contained therein.

[0190] In some embodiments, the nucleotide sequence of a gapmer may contain sufficient complementarity to an exon sequence. Petition 870250098454, dated 10 / 28 / 2025, pp. 91 / 170 84 / 141 within an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., a transcript encoding a protein of interest) or a region contained therein. In some embodiments, the nucleotide sequence of a gapmer may contain sufficient complementarity to an intron sequence within an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., a transcript encoding a protein of interest) or a region contained therein. In some embodiments, the nucleotide sequence of a gapmer may contain sufficient complementarity to a pre-mRNA transcript or an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., a transcript encoding a protein of interest) or a region contained therein.

[0191] For any of the methods described herein, different gapmers (e.g., two or more, three or more, four or more, or five or more different gapmers) can be combined to reduce the expression of a transgene of interest (e.g., a therapeutic transgene encoding a protein of interest; e.g., a transgene incorporated into an AAV vector described herein). A combination of two or more gapmers, such as two different gapmers, three different gapmers, four different gapmers, five or more different gapmers, may have overlapping regions of complementarity to an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript encoding a protein of interest) and may be used in a disclosure method to inhibit said transcript.Alternatively, two different gapmers, three different gapmers, four different gapmers, five or more different gapmers may not have any overlapping region of complementarity to an mRNA transcript (e.g., an mRNA transcript transcribed from the transgene of interest; e.g., an mRNA transcript). Petition 870250098454, dated 10 / 28 / 2025, page 92 / 170 85 / 141 which encodes a protein of interest) and are instead complementary to distinct regions of a transcript and can be used in a disclosure method to inhibit the mRNA transcript. G. Modifications in Nucleic Acid Molecules

[0192] It is contemplated that any of the nucleic acid molecules described herein (e.g., inhibitory nucleic acid molecules) may be used in the methods disclosed herein in an unmodified form or in a modified form. Unmodified nucleic acid molecules contain nucleobases which include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid molecules are described in more detail below.

[0193] Modifications can be achieved by systematically adding or removing linked nucleosides to generate longer or shorter sequences.

[0194] Modifications can be achieved by incorporating, for example, one or more alternative nucleosides, alternative 2' sugar moieties, and / or alternative internucleoside linkages. Typically, these types of modifications are introduced to optimize the efficacy or biophysical properties of the molecule (e.g., increase serum stability or circulating half-life, increase thermal stability, improve transmembrane administration, reduce immunogenicity, and / or target a specific site or cell type). By way of example, a modified nucleotide, such as a blocked nucleic acid (LNA), a peptide nucleic acid (PNA), or a bridged nucleic acid (BNA), can be incorporated into any nucleic acid-based inhibitor described above. Other nucleic acid modifications are described below.

[0195] The modification can also be achieved by covalent or non-covalent conjugation of a fraction (for example, a fraction of Petition 870250098454, dated 10 / 28 / 2025, page 93 / 170 86 / 141 targeting, a hydrophobic fraction, a cell-penetrating peptide or a polymer) to the 5' and / or 3' end of the inhibitory nucleic acid molecule, as described in more detail below. i. Nucleoside Modifications

[0196] The modification of inhibitory nucleic acid molecules described herein includes one or more of the following nucleoside modifications: 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine and other alkyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and others Adenines and guanines substituted in 8,5-halo, particularly 5-bromo, 5-trifluoromethyl and other uracils and cytosines substituted in 5,7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-aza-adenine, 7-desazaguanine and 7-desaz-adenine and / or 3-desazaguanine and 3-desa-azadenine.Inhibitory nucleic acid molecules may also include nucleobases in which the purine or pyrimidine base is replaced by other heterocycles, for example, 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, and / or 2-pyridone. Further modifications of the inhibitory nucleic acid molecules described herein may include nucleobases disclosed in US 3,687,808; Kroschwitz, JI, ed. The Concise Encyclopedia of Polymer Science and Engineering, New York, John Wiley & Sons, 1990, pp. 858-859; Englisch et al., Angewandte Chemie, International Edition 30: 613, 1991; and Sanghvi, YS, Chapter 16, Antisense Research and Applications, CRC Press, Gait, MJ ed., 1993, pp. 289-302. ii. Sugar Modifications

[0197] Modifications of the inhibitory nucleic acid molecules described herein may also include one or more of the following modifications. Petition 870250098454, dated 10 / 28 / 2025, p. 94 / 170 87 / 141 of 2' sugar: 2'-O-methyl (2'-O-Me), 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE), 2'-dimethylaminooxyethoxy, i.e., an O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, and / or 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2OCH2N(CH3)2. Other possible 2' modifications that may modify the inhibitory nucleic acid molecules described herein include all possible orientations of OH; F; O-, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Other potential sugar substituent groups include, for example, aminopropoxy (-OCH2CH2CH2NH2), allyl (-CH2-CH=CH2), -O-allyl (-O-CH2-CH=CH2), and fluorine (F). The 2'-sugar substituent groups may be in the arabino (upward) or ribo (downward) position.In some embodiments, the 2'-arabino modification is 2'-F. Similar modifications can also be made at other positions in the interfering RNA molecule, particularly at the 3' position of the sugar in the 3'-terminal nucleoside or in 2'-5' linked oligonucleotides and at the 5' position of the 5'-terminal nucleotide. The oligonucleotides may also have sugar mimetics, such as cyclobutyl moieties instead of the pentofuranosyl sugar. iii. Internucleoside Bond Modifications

[0198] Modifications of the inhibitory nucleic acid molecules described herein may include one or more of the following internucleoside modifications: phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates, 5'-alkylene phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranephosphates with normal 3'-5' linkages, 2'-5' linked analogs thereof and those with Petition 870250098454, dated 10 / 28 / 2025, page 95 / 170 88 / 141 reversed polarity, in which one or more internucleotide bonds are 3' to 3', 5' to 5', or 2' to 2' bonds. iv. Conjugates

[0199] Any of the inhibitory nucleic acid molecules described herein may be modified by the addition of an auxiliary moiety, for example, a cell-penetrating peptide (CPP), a polymer, a hydrophobic moiety, or a targeting moiety. The auxiliary moiety may be present as a 5'-terminal modification (e.g., covalently linked to a 5'-terminal nucleoside), a 3'-terminal modification (e.g., covalently linked to a 3'-terminal nucleoside), or an internucleoside linkage (e.g., covalently linked to phosphate or phosphorothioate in an internucleoside linkage).

[0200] CPPs are known in the art (e.g., TAT or Arg8) (Snyder and Dowdy, 2005, Expert Opin. Drug Deliv. 2, 43-51). Specific examples of CPPs are provided in WO2011157713, which is incorporated herein by reference in its entirety.

[0201] Dissemination-inhibiting nucleic acid molecules may include auxiliary moieties based on covalently linked neutral polymers. Neutral polymers include poly(C1-6 alkylene oxide), for example, poly(ethylene glycol) and poly(propylene glycol) and copolymers thereof, for example, di- and triblock copolymers.

[0202] An inhibitory nucleic acid molecule containing a hydrophobic moiety may exhibit superior cellular uptake and / or pharmacokinetic properties compared to an inhibitory nucleic acid molecule without the hydrophobic moiety. A hydrophobic moiety is a monovalent group (e.g., a bile acid; e.g., cholic acid, taurocholic acid, deoxycholic acid, oleyl lithocholic acid, or oleoyl cholenic acid), glycolipid, phospholipid, sphingolipid, isoprenoid, vitamin, fatty acid Petition 870250098454, dated 10 / 28 / 2025, pp. 96 / 170 89 / 141 saturated, unsaturated fatty acid, fatty acid ester, triglyceride, cholesterol, pyrene, porphyrin, texafirin, adamantine, acridine, biotin, coumarin, fluorescein, rhodamine, Texas-Red, digoxigenin, dimethoxytritil, t-butidimethylsilyl, t-butyldiphenylsilyl, cyanine dye (e.g., Cy3 or Cy5), Hoechst 33258 dye, psoralen or ibuprofen) covalently linked to the nucleic acid structure (e.g., 5' terminal) of the inhibitory nucleic acid molecule. In some embodiments, a hydrophobic moiety is cholesterol. III. Pharmaceutical Compositions

[0203] 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 in vivo administration. For example, the agents described herein may be administered in a suitable diluent, carrier, stabilizer, or excipient and may further contain a preservative, for example, to prevent the growth of microorganisms. Conventional procedures and ingredients for the selection and preparation of suitable compositions are described, for example, in Remington, JP 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.

[0204] Mixtures of agents described in this document 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 normal storage and use conditions, these preparations may contain a preservative to prevent the growth of microorganisms. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of solutions. Petition 870250098454, dated 10 / 28 / 2025, page 97 / 170 90 / 141 or sterile injectable dispersions (described in US 5,466,468, disclosure of which is incorporated herein by reference). In either case, the formulation may be sterile and may be flowable to the extent that easy syringeability exists. The formulations may be stable under manufacturing and storage conditions and may be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof and / or vegetable oils. Appropriate flowability may be maintained, for example, by the use of a coating, such as lecithin, by maintaining the necessary particle size in the case of dispersion and by the use of surfactants.The prevention of microbial action can be achieved 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 injectable compositions can be caused by the use of agents in the compositions that delay absorption, for example, gelatin and aluminum monostearate.

[0205] For example, a solution containing a pharmaceutical composition described in this document may be adequately buffered, if necessary, and the liquid diluent first made isotonic with sufficient saline or glucose solution. These specific aqueous solutions are particularly suitable for intramuscular, intravenous, subcutaneous, and intraperitoneal administration.

[0206] Although the descriptions of pharmaceutical compositions provided here are primarily directed to pharmaceutical compositions that are suitable for administration to humans, a qualified professional will understand that such compositions are generally suitable for administration. Petition 870250098454, dated 10 / 28 / 2025, page 98 / 170 91 / 141 to any other animal, for example, to non-human animals, for example, non-human mammals. The modification of pharmaceutical compositions suitable for administration to humans to make the compositions suitable for administration to various animals is well understood, and the veterinary pharmacologist with normal qualifications can design and / or perform such a modification with only ordinary experimentation, if any. The subjects to whom administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and / or other primates and mammals.

[0207] Compositions containing an agent such as a nucleic acid or an inhibitory nucleic acid molecule described herein may include a delivery vehicle for administration to a subject or to one or more of the subject's cells. Exemplary delivery vehicles for an inhibitory nucleic acid molecule described herein include, but are not limited to, lipid-based carriers, lipid nanoformulations such as lipid nanoparticles (LNPs), and suitable polymers.

[0208] Compositions containing an agent, such as a nucleic acid or vector described herein, may further include a second agent (for example, a nucleic acid molecule to be expressed within a cell, a polypeptide, or a drug). For example, a second agent could be a blood pressure medication, a steroid, an analgesic, or an immunosuppressant. IV. Treatment Methods

[0209] Any of the compositions, such as nucleic acid molecules (e.g., polynucleotides comprising transgenes, inhibitory nucleic acid molecules and inhibitory RNA molecules), vectors and AAV vectors (e.g., rAAV vectors) and any pharmaceutical compositions thereof, may be used in a method of treatment for a disease or condition in a subject in need thereof (e.g., a human subject). Petition 870250098454, dated 10 / 28 / 2025, page 99 / 170 92 / 141 In some modalities, a treatment method may be prophylactic treatment for a subject at risk of a disease or condition. In other modalities, a treatment method may reduce, reverse, improve, stabilize, or enhance a disease state or condition in a subject. In still other modalities, methods may be used to alleviate, improve, reduce, or reverse one or more clinical manifestations of a disease or condition.

[0210] In some modalities, the treatment method is directed toward treating a metabolic disorder, a blood disorder, a cardiovascular disorder, a neurological disorder, an eye 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 1 below. Table 1: Exemplary Diseases and Target Genes Disease(s) Gene(s) Achondroplasia FGFR3 Achromatopsia CNGA3, CNGB3, GNAT2, PDE6C, PDE6H, ACHM2, ACHM3 Acute Kidney Injury NFkappaB, AATF, p85alpha, FAS, elements of the apoptosis cascade (e.g., FASR, Caspase 2, 3, 4, 6, 7, 8, 9, 10, AKT, TNF alpha, IGF1, IGF1R, RIPK1), p53 Age-Related Macular Degeneration Abcr, CCL2, CC2, CP, Timp3, cathepsin D, VLDLR, CCR2, sFLTOl (VEGF / PlGF (placental growth factor) binding domain of human VEGFRI / Flt-1 (hVEGFRl) fused to the Fe portion of human IgG(l) via a ligand (of polyglycine) Acquired Immunodeficiency Syndrome (AIDS) KIR3DL1, NKAT3, NKB1, AMB11, KIR3DS1, IFNG, CXCL12, SDF1 Albinism (including oculocutaneous albinism (Types 1-7) and ocular albinism) TYR, OCA2, TYRP1, SLC45A2, SLC24A5, C10orf11 Alkaptonuria HGD Alpha-1 antitrypsin deficiency (AATD or AlAD) or hereditary emphysema AAT, SERPlNA1, those established in WO2017165862, PiZ allele Amyotrophic lateral sclerosis (ALS) SOD1, ALS2, ALS3, ALS5, ALS7, STEX,FUS, TARDBP, VEGF (VEGF-α; Petition 870250098454, dated 10 / 28 / 2025, pp. 100 / 170 93 / 141 Doença(s) Gene(s) VEGF-b; VEGF-c), DPP6, NEFH, PTGS1, SLC1A2, TNFRSF10B, PRPH, HSP90AA1, CRIA2, IFNG, AMPA2 S100B, FGF2, AOX1, CS, TXN, RAPHJ1, MAP3K5, NBEAL1, GPX1, ICA1L, RAC1, MAPT, ITPR2, ALS2CR4, GLS, ALS2CR8, CNTFR, ALS2CR11, FOLH1, FAM117B, P4HB, CNTF, SQSTM1, STRADB, NAIP, NLR, YWHAQ, SLC33A1, TRAK2, SCA1, NIF3L1, NIF3, PARD3B, COX8A, CDK15, HECW1, HECT, C2, WW 15, NOS1, MET, SOD2, HSPB1, NEFL, CTSB, ANG, HSPA8, RNase A, VAPB, VAMP, SNCA, alfa HGF, CAT, ACTB, NEFM, TH, BCL2, FAS, CASP3, CLU, SMN1, G6PD, BAX, HSF1, RNF19A, JUN, ALS2CR12, HSPA5, MAPK14, APEX1, TXNRD1, NOS2, TIMP1, CASP9, XIAP, GLG1, EPO, VEGFA, ELN, GDNF, NFE2L2, SLC6A3, HSPA4, APOE, PSMB8, DCTN2, TIMP3, KIFAP3, SLC1A1, SMN2, CCNC, STUB1, ALS2, PRDX6, SYP, CABIN1, CASP1, GART, CDK5, ATXN3, RTN4, C1QB, VEGFC, HTT, PARK7, XDH, GFAP, MAP2, CYCS, FCGR3B, CCS, UBL5, MMP9m SLC18A3, TRPM7, HSPB2, AKT1, DEERL1, CCL2, NGRN, GSR, TPPP3, APAF1, BTBD10, GLUD1, CXCR4, SLC1A3, FLT1, PON1, AR, LIF, ERBB3, GAS1, CD44, TP53, TLR3,GRIA1, GAPDH, AMPA, GRIK1, DES, CHAT, FLT4, CHMP2B, BAG1, CHRNA4, GSS, BAK1, KDR, GSTP1, OGG1, IL6 Alzheimer's disease E1, CHIP, UCH, UBB, Tau, LRP, PICALM, CLU, PS1, SORL1, CR1, VLDLR, UBA1, UBA3, CHIP28, AQP1, UCHL1, UCHL3, APP, AAA, CVAP, AD1, APOE, AD2, DCP1, ACE1, MPO, PACIP1, PAXIP1L, PTIP, A2M, BDNF, BLMH, BMH, PSEN1, AD3, ALAS2, ABCA1, BIN1, BDNF, BTNL8, C1ORF49, CDH4, CHRNB2, CKLFSF2,CLEC4E,CR1L,CSF3R, CST3, CYP2C, DAPK1, ESR1, FCAR, FCGR3B, FFA2, FGA, GAB2, GALP, GAPDHS, GDNF, GMPB, HP, HTR7, IDE, IF127, IFI6, IFIT2, IL1RN, IL-1RA, IL8RA, IL8RB, JAG1, KCNJ15, LRP6, MAPT, MARK4, MPHOSPH1, MTHFR, NBN, NCSTN, NIACR2, NGF, NMNAT3, NTM, ORM1, P2RY13, PBEF1, PCK1, PICALM, PLAU, PLXNC1, PRNP, PSEN1, PSEN2, PTPRA, RALGPS2, RGSL2, SELENBP1, SLC25A37, SORL1, Mitoferrina-1, TF, TFAM, TNF, TNFRSF10C, UBE1C Amyloidose APOA1, APP, AAA, CVAP, AD1, GSN, FGA, LYZ, TTR, PALB Neuropatia Amyloide TTR, PALB Anemia CDAN1, CDA1, RPS19, DBA, PKLR, PK1, NT5C3, UMPH1, PSN1, RHAG, RH50A, NRAMP2, SPTB,ALAS2, ANH1, ASB, ABCB7, ABC7, ASAT Angelman Syndrome UBE3A Anxiety BDNF Arthritis, Rheumatoid Arthritis TNFR:Fc, Petition 870250098454, dated 10 / 28 / 2025, pp. 101 / 170 94 / 141 Disease(s) Gene(s) Attention Deficit Hyperactivity Disorder (ADHD) PTCHD1 Autoimmune Lymphoproliferative Syndrome TNFRSF6, APT1, FAS, CD95, ALPS1A Autism, autism spectrum disorders (ASDs), including Asperger's syndrome and a general diagnostic category called pervasive developmental disorders (PDDs) PTCHD1, Mecp2, BZRAP1, MDGA2, Sema5A, Neurexin 1, GLO1, RTT, PPMX, MRX16, RX79, NLGN3, NLGN4, KIAA1260, AUTSX2, FMR1, FMR2, FXR1, FXR2, MGLUR5, ATP10C, CDH10, GRM6, MGLUR6, CDH9, CNTN4, NLGN2, CNTNAP2, SEMA5A, DHCR7, NLGN4X, NLGN4Y, DPP6, NLGN5, EN2, NRCAM, MDGA2, NRXN1, FMR2, AFF2, FOXP2, OR4M2, OXTR, FXR1, FXR2, PAH, GABRA1, PTEN, GABRA5, PTPRZ1, GABRB3, GABRG1, HIRIP3, SEZ6L2, HOXA1, SHANK3, IL6, SHBZRAP1, LAMB1, SLC6A4, SERT, MAPK3, TAS2R1, MAZ, TSC1, MDGA2, TSC2, MECP2, UBE3A, WNT2, see also 20110023145 Autosomal Dominant Polycystic Kidney Disease (ADPKD) - (includes diseases such as von Rippel-Lindau disease and...) Tuberous Sclerosis Complex PKD1,PKD2 Autosomal Recessive Polycystic Kidney Disease (ARPKR) PKDH1 Ataxia-Telangiectasia (also known as Louis Bar syndrome) ATM B-cell Non-Hodgkin Lymphoma BCL7A, BCL7 Bardet-Biedl Syndrome ARL6, BBS1, BBS2, BBS4, BBS5, BBS7, BBS9, BBS10, BBS12, CEP290, INPP5E, LZTFL1, MKKS, MKS1, SDCCAGS, TRIM32, TTC8 Nude Lymphocyte Syndrome TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11, MHC2TA, C2TA, RFX5, RFXAP, RFX5 Bartter Syndrome (Types I, II, III, IVA and B, and V) SLC12A1 (type I), KCNJ1 (type II), CLCNKB (type III), BSND (type IV A), or both genes CLCNKA CLCNKB (type IV B), CASR (type V) Batten disease, Late Infantile Neuronal Ceroid Lipofuscinosis CLN2 Becker Muscular Dystrophy DMD, BMD, MYF6 Best disease (Vitelliform Macular Dystrophy Type 2) VMD2 Bipolar Disorder BDNF Bleeding Disorders TBXA2R, P2RX1, P2X1 Blue Cone Monochromacy OPN1LW, OPN1MW, LCR Breast Cancer BRCA1, BRCA2,COX-2 Bruton's disease (also known as X-linked agammaglobulinemia) BTK Canavan's disease ASPA Cancers (e.g., lymphoma, chronic lymphocytic leukemia (CLL), acute lymphocytic B-cell leukemia (A-ALL), acute lymphoblastic leukemia, FAS leukemia, BID, CTLA4, PDCD1, CBLB, PTPN6, TRAC, TRBC, those described in WO2015048577, Petition 870250098454, dated 10 / 28 / 2025, page 102 / 170 95 / 141 Illnesses) Acute myeloid lymphoma, non-Hodgkin lymphoma (NHL), diffuse large cell lymphoma (DLCL), multiple myeloma, renal cell carcinoma (RCC), neuroblastoma, colorectal cancer, breast cancer, ovarian cancer, melanoma, sarcoma, lung cancer, esophageal cancer, hepatocellular carcinoma, pancreatic cancer, astrocytoma, mesothelioma, head and neck cancer, and medulloblastoma. Cardiovascular Diseases IL1B, XDH, TP53, PTGS, MB, IL4, ANGPT1, ABCGu8, CTSK, PTGIR, KCNJ11, INS, CRP, PDGFRB, CCNA2, PDGFB, KCNJ5, KCNN3, CAPN10, ADRA2B, ABCG5, PRDX2, CPAN5, PARP14, MEX3C, ACE, RNF, IL6, TNF, STN, SERPINE1, ALB, ADIPOQ, APOB, APOE, LEP, MTHFR, APOA1, EDN1, NPPB, NOS3, PPARG, PLAT, PTGS2, CETP, AGTR1, HMGCR, IGF1, SELE, REN, PPARA, PON1, KNG1, CCL2, LPL, VWF, F2, ICAM1, TGFB NPPA, IL10, EPO, SOD1, VCAM1, IFNG, LPA, MPO, ESR1, MAPK, HP, F3, CST3, COG2, MMP9, SERPINC1, F8, HMOX1, APOC3, IL8, PROL1, CBS, NOS2, TLR4, SELP, ABCA1, AGT, LDLR, GPT, VEGFA, NR3C2, IL18, NOS1, NR3C1, FGB, HGF, IL1A, AKT1, LIPC, HSPD1, MAPK14, SPP1, ITGB3, CAT, UTS2, THBD, F10, CP, TNFRSF11B, EGFR, MMP2, PLG, NPY, RHOD, MAPK8, MYC, FN1, CMA1, PLAU, GNB3, ADRB2, SOD2, F5, VDR, ALOX5, HLADRB1, PARP1, CD40LG, PON2, AGER, IRS1, PTGS1, ECE1, F7, IRMN, EPHX2, IGFBP1, MAPK10, FAS, ABCB1, JUN, IGFBP3, CD14, PDE5A, AGTR2, CD40, LCAT, CCR5, MMP1, TIMP1, ADM, DYT10, STAT3, MMP3, ELN, USF1, CFH, HSPA4, MMP12, MME, F2R, SELL, CTSB,ANXA5, ADRB1, CYBA, FGA, GGT1, LIPG, HIF1A, CXCR4, PROC, SCARB1, CD79A, PLTP, ADD1, FGG, SAA1, KCNH2, DPP4, NPR1, VTN, KIAA0101, FOS, TLR2, PPIG, IL1R1, AR, CYP1A1, SERPINA1, MTR, RBP4, APOA4, CDKN2A, FGF2, EDNRB, ITGA2, VLA-2, CABIN1, SHBG, HMGB1, HSP90B2P, CYP3A4, GJA1, CAV1, ESR2, LTA, GDF15, BDNF, CYP2D6, NGF, SP1, TGIF1, SRC, EGF, PIK3CG, HLA-A, KCNQ1, CNR1, FBN1, CHKA, BEST1, CTNNB1, IL2, CD36, PRKAB1, TPO, ALDH7A1, CX3CR1, TH, F9, CH1, TF, HFE, IL17A, PTEN, GSTM1, DMD, GATA4, F13A1, TTR, FABP4, PON3, APOC1, INSR, TNFRSF1B, HTR2A, CSF3, CYP2C9, TXN, CYP11B2, PTH, CSF2, KDR, PLA2G2A, THBS1, GCG, RHOA, ALDH2, TCF7L2, NFE2L2, NOTCH1, UGT1A1, IFNA1, PPARD, SIRT1, GNHR1, PAPPA, ARR3, NPPC, AHSP,PTK2, IL13, MTOR, ITGB2, GSTT1, IL6ST, CPB2, CYP1A2, HNF4A, SLC64A, PLA2G6, TNFSF11, SLC8A1, F2RL1, AKR1A1, ALDH9A1, BGLAP, MTTP, MTRR, SULT1A3, RAGE, C4B, P2RY12, Petition 870250098454, on 10 / 28 / 2025, page. 103 / 170 96 / 141 Doença(s) Gene(s) RNLS, CREB1, POMC, RAC1, LMNA, CD59, SCM5A, CYP1B1, MIF, MMP13, TIMP2, CYP19A1, CUP21A2, PTPN22, MYH14, MBL2, SELPLG, AOC3, CTSL1, PCNA, IGF2, ITGB1, CAST, CXCL12, IGHE, KCNE1, TFRC, COL1A1, COL1A2, IL2RB, PLA2G10, ANGPT2, PROCR, NOX4, HAMP, PTPN11, SLCA1, IL2RA, CCL5, IRF1, CFAR, CACA, EIF4E, GSTP1, JAK2, CYP3A5, HSPG2, CCL3, MYDSS, VIP, SOAT1, ADRBK1, NR4A2, MMPS, NPR2, GCH1, EPRS, PPARGC1A, F12, PECAM1, CCL4, CERPINA34, CASR, FABP2, TTF2, PROS1, CTF1, SGCB, YME1L1, CAMP, ZC3H12A, AKR1B1, MMP7, AHR, CSF1, HDAC9, CTGF, KCNMA1, UGT1A, PRKCA, COMT, S100B, EGR1, PRL, IL15, DRD4, CAMK2G, SLC22A2, CCL11, PGF, THPO, GP6, TACR1, NTS, HNF1A, SST, KCDN1, LOC646627, TBXAS1, CUP2J2, TBXA2R, ADH1C, ALOX12, AHSG, BHMT, GJA4, SLC25A4, ACLY, ALOX5AP, NUMA1, CYP27B1, CYSLTR2, SOD3, LTC4S, UCN, GHRL, APOC2, CLEC4A, KBTBD10, TNC, TYMS, SHC1, LRP1, SOCS3, ADH1B, KLK3, HSD11B1, VKORC1, SERPINB2, TNS1, RNF19A, EPOR, ITGAM, PITX2, MAPK7, FCGR3A, LEEPR, ENG, GPX1, GOT2, HRH1, NR112, CRH,HTR1A, VDAC1, HPSE, SFTPD, TAP2, RMF123, PTK2Bm NTRK2, IL6R, ACHE, GLP1R, GHR, GSR, NQO1, NR5A1, GJB2, SLC9A1, MAOA, PCSK9, FCGR2A, SERPINF1, EDN3, UCP2, TFAP2A, C4BPA, SERPINF2, TYMP, ALPP, CXCR2, SLC3A3, ABCG2, ADA, JAK3, HSPA1A, FASN, FGF1, F11, ATP7A, CR1, GFPA, ROCK1, MECP2, MYLK, BCHE, LIPE, ADORA1, WRN, CXCR3, CD81, SMAD7, LAMC2, MAP3K5, CHGA, IAPP, RHO ENPP1, PTHLH, NRG1, VEGFC, ENPEP, CEBPB, NAGLU, F2RL3, CX3CL1, BDKRB1, ADAMTS13, ELANE, ENPP2, CISH, GAST, MYOC, ATP1A2, NF1, GJB1, MEF2A, VCL, BMPR2, TUBB, CDC42, KRT18, HSF1, MYB, PRKAA2, ROCK2, TFP1, PRKG1, BMP2, CTNND1, CTH, CTSS, VAV2, NPY2R, IGFBP2, CD28, GSTA1, PPIA, APOH, S100A8, IL11, ALOX15, FBLN1, NR1H3, SCD, GIP, CHGB, PRKCB, SRD5A1, HSD11B2,CALCRL, GALNT2, ANGPTL4, KCNN4, PIK3C2A, HBEGF, CYP7A1, HLA-DRB5, BNIP3, GCKR, S100A12, PADI4, HSPA14, CXCR1, H19, KRTAP19-3, IDDM2, RAC2, YRY1, CLOCK, NGFR, DBH, CHRNA4, CACNA1C, PRKAG2, CHAT, PTGDS, NR1H2, TEK, VEGFB, MEF2C, MAPKAPK2, TNFRSF11A, HSPA9, CYSLTR1, MAT1A, OPRL1,IMPA1, CLCN2, DLD, PSMA6, PSMBS, CHI3L1, ALDH1B1, PARP2, STAR, LBP, ABCC6, RGS2, EFNB2, GJB6, APOA2, AMPD1, DYSF, FDFT1, EMD2, CCR6, GJB3, IL1RL1, ENTPD1, ​​BBS4, CELSR2, F11R, RAPGEF3, HYAL1, ZNF259, ATOX1, ATF6, KHK, SAT1, GGH, TIMP4, SLC4A4, PDE2A, Petition 870250098454, dated 10 / 28 / 2025, pp. 104 / 170 97 / 141 Disease Gene(s) PDE3B, FADS1, FADS2, TMSB4X, TXNIP, LIMS1, RHOB, LY96, FOXO1, PNPLA2, TRH, GJC1, SLC7A5, FTO, GJD2, PRSC1, CASP12, GPBAR1, PXK, IL33, TRIB1, PBX4, NUPR1, 15-SEP, CILP2, TERC, GGT2, MTCO1, UOX, AVP, ANGPLT3 Cataract CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2, CP49, CP47, CRYAA, PAX6, AN2, MGDA, CRYBA1, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19, CRYGD, CRYG4, BFSP2, CP49, CP47, HSF4, CTM, MIP, AQP0, CRYAB, CRYA2, CTPP2, CRYBB1, CRYGD, CRYG4, CRYBB2, CRYB2, CCL, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1 Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) CASQ2 CDKL-5 Deficiencies or CDKL5-Mediated Diseases Charcot-Marie-Tooth Disease (CMT) (Types 1, 2, 3 and 4) PMP22 (CMT1A and E), MPZ (CMT1B), LITAF (CMT1C), EGR2 (CMT1D), NEFL (CMT1F), GJB1 (CMT1X), MFN2 (CMT2A), KIF1B (CMT2A2B), RAB7A (CMT2B), TRPV4 (CMT2C), GARS (CMT2D), NEFL (CMT2E), GAPD1 (CMT2K), HSPB8 (CMT2L), DYNC1H1 (CMT2O), LRSAM1 (CMT2P), IGHMBP2 (CMT2S), MORC2 (CMT2Z)MTMR2 or SBF2 / MTMR13 (CMT4B), SH3TC2 (CMT4C), NDRG1 (CMT4D), PRX (CMT4F), FIG4 (CMT4J), NT-3 Chediak-Higashi Syndrome LYST Choroideremia CHM, REP1 Chorioretinal Atrophy PRDM13, RGR, TEAD1 Chronic Granulomatous Disease CYBA, CYBB, NCF1, NCF2, NCF4 Chronic Heart Failure SERCA2 Chronic Mucocutaneous Candidiasis AIRE, CARD9, CLEC7A IL12B, IL12B1, IL1F, IL17RA, IL17RC, RORC, STAT1, STAT3, TRAF31P2 Cirrhosis KRT18, KRT8, CIRH1A, NAIC, TEX292, KIAA1988 Colon Cancer (familial adenomatous polyposis) (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC) FAP: APC, HNPCC: MSH2, MLH1, PMS2, SH6, PMS1 Combined Immunodeficiency IL2RG, SCIDX1, SCIDX, IMD4, HIV-1 (CCL5, SCYA5, D17S136E, TCP228) Cone-rod dystrophy AIPL1, CRX, GUA1A, GUCY2D, PITPM3, PROM1, PRPH2, RIMS1, SEMA4A, ABCA4, ADAM9, ATF6, C21ORF2, C8ORF37, CACNA2D4, CDHR1, CERKL, CNGA3, CNGB3, CNNM4, CNAT2, IFT81, KCNV2, PDE6C, PDE6H, POC1B, RAX2, RDH5, RPGRIP1, TTLL5, RetCG1,GUCY2E Congenital Stationary Night Blindness CABP4, CACNA1F, CACNA2D4, GNAT1, CPR179, GRK1, GRM6, LRIT3, NYX, PDE6B, RDH5, RHO, RLBP1, RPE65, SAG, SLC24A1, TRPM1, Petition 870250098454, dated 10 / 28 / 2025, pp. 105 / 170 98 / 141 Disease(s) Gene(s) Congenital Fructose Intolerance ALDOB Cori Disease (Glycogen Storage Disease Type III) AGL Corneal opacity and dystrophy APOA1, TGFB1, CSD2, CDGG1, CSD, BIGH3, CDG2, TACSTD2, TROP2, M1S1, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD, PPCD2, PIP5K3, CFD Congenital flat cornea KERA, CNA2 Cri du Chat syndrome, also known as 5p syndrome and Cat Cry Syndrome Deletions involving only the 5p15 band.2 on the entire short arm of chromosome 5, for example, CTNND2, TERT Crigler-Najjar Syndrome UGT1A1 Cystic Fibrosis (CF) CTFR, ABCC7, CF, MRP7, SCNN1A, those described in WO2015157070 Diabetic Nephropathy Gremlin, 12 / 15-lipoxygenase, TIM44 Dental Disease (types 1 and 2) Type 1: CLCN5, Type 2: ORCL Dentatorrubro-Palidoluysian Atrophy (DRPLA) (also known as Haw River Disease and Naito-Oyanagi Disease) Atrophin-1 and ATN1 Down Syndrome Trisomy 21 Drug Dependence PRKCE, DRD2, DRD4, ABAT, GRIA2, GRM5, GRIN1, HTR1b, GRIN2A, DRD3, PDYN, GRIA1 Duane syndrome (Types 1, 2 and 3, including subgroups A, B and C).Other names for this condition include Duane Retraction Syndrome (or DR Syndrome), Ocular Retraction Syndrome, Retraction Syndrome, Congenital Retraction Syndrome, and Stilling-Turk-Duane Syndrome. CHN1, indels on chromosomes 4 and 8. Duchenne Muscular Dystrophy (DMD). DMD, BMD, dystrophin gene, intron flanking exon 51 of the DMD gene, exon 51 mutations in the DMD gene, see also WO2013163628 and Pub. Pt.US 20130145487 Eating Disorder BDNF Edwards Syndrome (Trisomy 18) Complete or partial trisomy of chromosome 18 Ehlers-Danlos Syndrome (Types I-VI) COL5A1, COL5A2, COL1A1, COL3A1, TNXB, PLOD1, COL1A2, FKBP14, ADAMTS2 Emery-Dreifuss Muscular Dystrophy LMNA, LMN1, EMD2, FPLD, CMD1A, HGPS, LGMD1B, LMNA, LMN1, EMD2, FPLD, CMD1A Enhanced Cone Syndrome NR2E3, NRL Epilepsy NPY Fabry Disease GLA, AGA Facioscapulohumeral Muscular Dystrophy FSHMD1A, FSHD1A, FRG1 Factor H and Factor T H 1 HF1, CFH, HUS Factor V Leiden Thrombophilia and Factor V Deficiency Factor V (F5) Factor V and Factor VII Deficiency MCFD2. Petition 870250098454, dated 10 / 28 / 2025, pp. 106 / 170 99 / 141 Disease(s) Gene(s) Factor VII Deficiency F7 Factor X Deficiency F10 Factor XI Deficiency F11 Factor XII Deficiency F12, HAF Factor XIIIA Deficiency F13A1, F13A Factor XIIIB Deficiency F13B Familial Hypercholesterolemia APOB, LDLR, PCSK9 Familial Mediterranean Fever (FMF), also called Recurrent Polyserositis or Familial Paroxysmal Polyserositis MEFV Fanconi Anemia FANCA, FACA, FA1, FA, FAA, FAAP95, FAAP90, FLJ34064, FANCC, FANCG, RAD51, BRCA1, BRCA2, BRIP1, BACH1, FANCJ, FANCB, FANCD1, FANCD2, FANCD, FAD, FANCE, FACE, FANCF, FANCI, ERCC4, FANCL, FANCM, PALB2, RAD51C, SLX4, UBE2T, FANCB, XRCC9, PHF9, KIAA1596 Fanconi Syndrome Types I (Childhood onset) and II (Adult-onset) FRTS1, GATM Fragile X Syndrome and related disorders FMR1, FMR2, FXR1, FXR2, mGLUR5 Mental Retardation Fragile XE (also known as Martin-Bell Syndrome) FMR1 Friedreich's Ataxia (FRDA) FXN / X25 Fuchs' Corneal Endothelial Dystrophy TCF4, COL8A2 Galactosemia GALT,GALK1, GALE Gastrointestinal epithelial cancer, GI cancer CISH Gaucher disease (types 1, 2 and 3, as well as other uncommon forms that may not fit into these types) GBA, GCase Glaucoma MYOC, TIGR, GLC1A, JOAG, GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1B1, GLC3A, OPA1, NTG, NPG, CYP1B1, GLC3A, those described in WO2015153780 Glomerulosclerosis CC chemokine ligand 2 Glycogen storage diseases (types I-VI - see also Cori disease, Pompe disease, McArdle disease, Hers disease and Von Gierke disease) SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GBE1, GYS2, PYGL, PFKM, see also Cori disease, Pompe disease, McArdle disease, Hers disease and Von Gierke disease. Glycolytic enzyme deficiency in erythrocytes: any mutations in a gene for an enzyme in the glycolysis pathway, including mutations in genes for hexokinases I and II, glucokinase, phosphoglucose isomerase, phosphofructokinase, aldolase, Bm triosephosphate isomerase.Glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerokinase, phosphoglycerate mutase, enolase I, pyruvate kinase. GM2 gangliosidoses (e.g., Sandhoff disease and Tay disease) - Hexosaminidase-alpha, hexosaminidase-beta, HEXA, Petition 870250098454, dated 10 / 28 / 2025, pp. 107 / 170 100 / 141 Disease(s) Gene(s) Sachs) Glycogen Storage Diseases type Ia (GSDIa) G6Pase Hemophilia (A or B) F8, F9 Hartnup Disease SLC6A19 Hearing Loss NOX3, Hes5, BDNF Hemochromatosis (HH) HFE, H63D Hemophagocytic Lymphohistiocytosis Disorders PRF1, HPLH2, UNC13D, MUNC13-4, HPLH3, HLH3, FHL3 Bleeding Disorders PI, ATT, F5 Hers Disease (Glycogen Storage Disease Type VI) PYGL Hereditary Angioedema (HAE) Kallikrein B1 Hereditary Hemorrhagic Telangiectasia (Osler-Weber-Rendu Syndrome) ACVRL1, ENG, SMAD4 Hereditary Spherocytosis NK1, EPB42, SLC4A1, SPTA1, SPTB Hereditary Persistence of Fetal Hemoglobin HBG1, HBG2, BCL11A, HBG 1 and / or 2 promoter region (in the CCAAT box) Hemophilia (Hemophilia A (Classic), B (also known as Natal disease) and C) A: FVIII, F8C, HEMA B: FVIX, HEME, FIX C: F9, F11 Hepatic Adenoma TCF1, HNF1A, MODY3 Liver failure, early onset and neurological disorder SCOD1,SCO1 Hepatic Lipase Deficiency LIPC Hepatoblastoma, Cancer and Carcinomas CTNNB1, PDGFRL, PDGRL, PRLTS, AXIN1, AXIN, CTNNB1, TP53, P53, LFS1, IGF2R, MPR1, MET, CASP8, MCH5 Hermansky-Pudlak Syndrome HPS1, HPS3, HPS4, HPS5, HPS6, HPS7, DTNBP1, BLOC1, BLOC1S2, BLOC3 Susceptibility or infection with human immunodeficiency virus (HIV) IL10, CSIF, CMKBR2, CCR2, CMKBR5, CCCKR5 (CCR5), those in WO2015148670A1 Holoprosencephaly (HPE) (Alobar, Semilobar and Lobar) ACVRL1, ENG, SMAD4 Homocystinuria CBS, MTHFR, MTR, MTRR, MMADHC Human papillomavirus (HPV) HPV16, HPV18 E6 / E7 Herpes simplex virus (HSV)1, HSV2 and related keratitis HSV1 genes (immediate early and late HSV-1 genes (UL1, 1.5, 5, 6, 8, 9, 12, 15, 16, 18, 19, 22, 23, 26, 26.5, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 42, 48, 49.5, 50, 52, 54, S6, RL2, RS1, those described in WO2015153789,WO2015153791 Hunter syndrome (also known as Mucopolysaccharidosis Type II) IDS Huntington's disease (HD) and HD-like disorders HD, HTT, IT15, PRNP, PRIP, JPH3, JP3, HDL2, TBP, SCA17, PRKCE, BDNF, IGF1, EP300, RCOR1, PRKCZ, HDAC4, TGM2, those described in WO2013130824, WO2015089354, Petition 870250098454, dated 10 / 28 / 2025, pp. 108 / 170 101 / 141 Disease(s) Gene(s) Hurler Syndrome (also known as Mucopolysaccharidosis (MPS) Type IH, MPS IH) IDUA, α-L-iduronidase Hurler Syndrome (also known as Mucopolysaccharidosis Type IH-S, MPS IH-S) IDUA, α-L-iduronidase Hyaluronidase deficiency (also known as MPS IX) HYAL1 Hypercholesterolemia or Phenylketonuria (PKU) PAH, LDLR Hyper IgM Syndrome CD40L Hypertension caused kidney damage Mineral corticosteroid receptor Immunodeficiencies CD3E, CD3G, AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSF5, CD40LG, HIGM1, IGM, FOXP3, IPEX, AIID, XPID, PIDX, TNFRSF14B, TACI Inborn errors of metabolism: including urea cycle disorders, organic acidemias, fatty acid oxidation defects, aminoacidopathies, carbohydrate disorders, mitochondrial disorders See also: Carbohydrate metabolism disorders (e.g., galactosemia), Amino acid metabolism disorders (e.g., phenylketonuria), Fatty acid metabolism (e.g.,MCAD deficiency), Urea cycle disorders (e.g., citrullinemia), Organic acidemias (e.g., maple syrup urine disease), Mitochondrial disorders (e.g., MELAS), Peroxisomal disorders (e.g., Zellweger syndrome) Inflammation IL-10, IL-1 (IL-1a, IL-1b), IL-13, IL-17 (IL-17a (CTLA8), IL-17b, IL-17c, IL-17d, IL-17f), IL-23, Cx3crl, ptpn22, TNFα, NOD2 / CARD15 for IBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3c11 Inflammatory bowel diseases (e.g., ulcerative colitis and Crohn's disease) NOD2, IRGM, LRRK2, ATG5, ATG16L1, IRGM, GATM, ECM1, CDH1, LAMB1, HNF4A, GNA12, IL10, CARD9 / 15, CCR6, IL2RA, MST1, TNFSF15, REL, STAT3, IL23R, IL12B, FUT2 Interstitial Renal Fibrosis TGF-β type II receptor Job's syndrome (also known as Hyper IgE syndrome) STAT3, DOCK8 Juvenile Retinoschisis RS1, XLRS1 Kabuki syndrome 1 MLL4,KMT2D Kennedy disease (also known as Spinobulbar Muscular Atrophy) SBMA / SMAX1 / AR Klinefelter syndrome Extra X chromosome in males Lafora disease EMP2A, EMP2B Leber Congenital Amaurosis CRB1, RP12, CORD2, CRD, CRX, IMPDH1, OTX2, AIPL1, CABP4, CCT2, CEP290, CLUAP1, CRB1, CRX, DTHD1, GDF6, GUCY2D, IFT140, IQCB1, KCNJ13, LCA5, LRAT, NMNAT1, PRPH2, RD3, RDH12, RPE65, RP20, RPGRIP1, SPATA7, TULP1, LCA1, LCA4, GUC2D, CORD6, LCA3, Petition 870250098454, dated 10 / 28 / 2025, pp. 109 / 170 102 / 141 Doença(s) Gene(s) Óptica Hereditária de Leber ND4 Lesch-Nyhan Syndrome HPRT1 Leukocyte Deficiencies and Disorders ITGB2, CD18, LCAMB, LAD, EIF2B1, EIF2BA, EIF2B2, EIF2B3, EIF2B5, LVWM, CACH, CLE, EIF2B4 Leukemia TAL1, TCL5, SCL, TAL2, FLT3, NBS1, NBS, ZNFN1A1, IK1, LYF1, HOXD4, HOX4B, BCR, CML, PHL, ALL, ARNT, KRAS2, RASK2, GMPS, AF10, ARHGEF12, LARG, KIAA0382, CALM, CLTH, CEBPA, CEBP, CHIC2, BTL, FLT3, KIT, PBT, LPP, NPM1, NUP214, D9S46E, CAN, CAIN, RUNX1, CBFA2, AML1, WHSC1L1, NSD3, FLT3, AF1Q, NPM1, NUMA1, ZNF145, PLZF, PML, MYL, STAT5B, AF10, CALM, CLTH, ARL11, ARLTS1, P2RX7, P2X7, BCR, CML, PHL, ALL, GRAF, NF1, VRNF, WSS, NFNS, PTPN11, PTP2C, SHP2, NS1, BCL2, CCND1, PRAD1, BCL1, TCRA, GATA1, GF1, ERYF1, NFE1, ABL1, NQO1, DIA4, NMOR1, NUP214, D9S46E, CAN,CAIN Muscular dystrophies of the shoulder girdle LGMD Lipoprotein lipase (LPL) deficiency LPL Lowe syndrome OCRL Lupus glomerulonephritis MAPK1 Lysosomal storage disorders GDNF Machado-Joseph disease (also known as Spinocerebellar Ataxia Type 3) ATX3 Macular degeneration ABC4, CBC1, CHM1, APOE, C1QTNF5, C2, C3, CCL2, CCR2, CD36, CFB, CFH, CFHR1, CFHR3, CNGB3, CP, CRP, CST3, CTSD, CX3CR1, ELOVL4, ERCC6, FBLN5, FBLN6, FSCN2, HMCN1, HTRA1, IL6, IL8, PLEKHA1, PROM1, PRPH2, RPGR, SERPING1, TCOF1, TIMP3, TLR3 Macular Dystrophy BEST1, C1QTNF5, CTNNA1, EFEMP1, ELOVL4, FSCN2, GUCA1B, HMCN1, IMPG1, OTX2, PRDM13, PROM1, PRPH2, RP1L1, TIMP3, ABCA4, CFH, DRAM2, IMG1, MFSD8, ADMD, STGD2, STGD3, RDS, RP7, PRPH, AVMD, AOFMD, VMD2 Malattia Leventinese EFEMP1, FBLN3 Malignant Melanoma CD86, B7-2, IL-12 Maple Syrup Urine Disease BCKDHA, BCKDHB,DBT Marfan syndrome FBN1 Maroteaux-Lamy syndrome (also known as MPS VI) ARSB medium-chain acyl-CoA dehydrogenase (MCAD) deficiency MCAD McArdle disease (PYGM disease, Petition 870250098454, dated 10 / 28 / 2025, pp. 110 / 170 103 / 141 Disease(s) Gene(s) Glycogen Storage Disease Type V, myophosphorylase deficiency) Polycystic kidney disease UMOD, HNFJ, FJHN, MCKD2, ADMCKD2 Metachromatic Leukodystrophy ARSA Methylmalonic acidemia (MMA) MMAA, MMAB, MUT, MMACHC, MMADHC, LMBRD1 Mitochondrial heteroplasmy, Myoclonic epilepsy with irregular red fibers (MERRF) or Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) APALI Morquio syndrome (also known as MPS IVA and B) GALNS Mucopolysaccharidosis Type VII (MPSVII) or Sly syndrome GUSB Mucopolysaccharidosis diseases (Types IH / S, IH, II, III AB and C, IS, IVA and B, IX, VII and VI) See also Hurler / Scheie Syndrome, Hurler Disease, Sanfilippo Syndrome, Scheie Syndrome, Morquio Syndrome, Hyaluronidase Deficiency, Sly Syndrome and Maroteaux-Lamy Syndrome Muscular Atrophy VAPB, VAPC, ALS8, SMN1, SMA1, SMA2, SMA3, SMA4, BSCL2, SPG17, GARS, SMAD1, CMT2D, HEXB, IGHMBP2, SMUBP2, CATF1,SMARD1 Muscular Dystrophy Sarcoglycan α, β, γ, Δ, ε or ζ (SGCA, SGCB, SGCG, SGCD, SGCE or SGCZ), FKRP, MDC1C, LGMD2I, LAMA2, LAMM, LARGE, KIAA0609, MDC1D, FCMD, TTID, MYOT, CAPN3, CANP3, DYSF, LGMD2B, LGMD2C, DMDA1, SCG3, ADL, DAG2, LGMD2D, DMDA2, LGMD2E, SGD, LGMD2F, CMD1L, TCAP, LGMD2G, CMD1N, TRIM32, HT2A, LGMD2H, FKRP, MDC1C, LGMD2I, TTN, CMD1G, TMD, LGMD2J, POMT1, CAV3, LGMD1C, SEPN1, SELN, RSMD1, PLEC1, PLTN, EBS1, dystrophin, mini-dystrophin, Myotonic Dystrophy (Type 1 and Type 2), CNBP (Type 2), DMPK (Type 1), Neoplasia, PTEN, ATM, ATR, EGFR, ERBB2, ERBB3, ERBB4, NOTCH1, NOTCH2, NOTCH3, NOTCH4, AKT, AKT2, AKT3, HIF, HIF1A, HIF3A, MET, HRG, BCL2, PPAR alpha, PPAR gamma, WT1 (Wilms' tumor), FGF receptor family members (5 members: 1, 2, 3, 4, 5), CDKN2A, APC, RB (retinoblastoma), MEN1, VHL, BRCA1, BRCA2, Ar (androgen receptor), TSG101, IGF, receptor IGF, IGF1 (4 variants), IGF2 (3 variants), IGF1 receptor, IGF2 receptor, BAX, BCL2,Caspase family (9 members: 1, 2, 3, 4, 6, 7, 8, 9, 12), KRAS, APC Neurofibromatosis (NF) (NF1, formerly Recklinghausen's NF and NF2) NF1, NF2 Niemann-Pick Lipidosis (Types A, B and C) Types A and B: SMPD1, Type C: NPC1 or NPC2 Noonan Syndrome PTPN11, SOS1, RAF1, KRAS, Petition 870250098454, dated 10 / 28 / 2025, pp. 111 / 170 104 / 141 Disease(s) Gene(s) Norrie Disease or X-linked Familial Exudative Vitreoretinopathy NDP North Carolina Macular Dystrophy MCDR1 Ornithine carbamoyltransferase deficiency No prescription Osteogenesis Imperfecta (OI) (Types I, II, III, IV, V, VI and VII) COL1A1, COL1A2, CRTAP, P3H Osteopetrosis LRP5, BMND1, LRP7, LR3, OPPG, VBCH2, CLCN7, CLC7, OPTA2, OSTM1, GL, TCIRG1, TIRC7, OC116, OPTB1 Patau Syndrome (Trisomy 13) Extra copy of chromosome 13 Parkinson's Disease (PD) SNCA (PARK1), UCHL1 (PARK 5), and LRRK2 (PARK8), PARK3, PARK2, PARK4, PARK7, PINK1 (PARK6) RDS / peripheral Phenylketonuria (PKU) HAP, PKU1, QDPR, DHPR, PTS Polycystic Kidney and Liver Disease FCYT, PKHD1, ARPKD, PKD1, PKD2, PKD4, PKDTS, PRKCSH, G19P1, PCLD, SEC63 Pompe Disease GAA Porphyria (actually,This refers to a group of different diseases, all with a specific abnormality in the heme production process) ALAD, ALAS2, CPOX, FECH, HMBS, PPOX, UROD, UROS Posterior Polymorphic Corneal Dystrophy TCF4, COL8A2 Prader-Willi Syndrome Short arm region deletion of chromosome 15, including UBE3A Primary hyperoxaluria (e.g., type 1) LDHA (lactate dehydrogenase A) and hydroxyacid oxidase 1 (HAO1) ​​Primary open-angle glaucoma (POAG) MYOC Primary Sclerosing Cholangitis TCF4, COL8A2 Progeria (also called Hutchinson-Gilford Progeria Syndrome) LMNA Progressive external ophthalmoplegia ANT-I Propionic acidemia PCCA Prostate cancer HOXB13, MSMB, GPRC6A, TP53, GM-CSF Pyruvate Dehydrogenase Deficiency PDHA1 Kidney / Renal Carcinoma RLIP76, VEGF Rett Syndrome MECP2, RTT, PPMX, MRX16, MRX79, CDKL5, STK9, MECP2, RTT, PPMX, MRX16, MRX79, x-Synuclein, DJ-1 Retinitis pigmentosa (RP) ADIPOR1, ABCA4, AGBL5, ARHGEF18, ARL2BP, ARL3, ARL6, BEST1, BBS1, BBS2,C2ORF71, C8ORF37, CA4, CERKL, CLRN1, CNGA1, CMGB1, CRB1, CRX, CYP4V2, DHDDS, DHX38, Petition 870250098454, dated 10 / 28 / 2025, pp. 112 / 170 105 / 141 Disease Gene(s) EMC1, EYS, FAM161A, FSCN2, GPR125, GUCA1B, HK1, HPRPF3, HGSNAT, IDH3B, IMPDH1, IMPG2, IFT140, IFTl72, KLHL7, KIAA1549, KIZ, LRAT, MAK, MERTK, MVK, NEK2, NUROD1, NR2E3, NRL, OFD1, PDE6A, PDE6B, PDE6G, POMGNT1, PRCD, PROM1, PRPF3, PRPF4, PRPF6, PRPF8, PRPF31, PRPH2, RPB3, RDH12, REEP6, RP39, RGR, RHO, RLBP1, ROM1, RP1, RP1L1, RPY, RP2, RP9, RPE65, RPGR, SAMD11, SAG, SEMA4A, SLC7A14, SNRNP200, SPP2, SPATA7, TRNT1, TOPORS, TTC8, TULP1, USH2A, ZNF408, ZNF513, see also 20120204282 Sanfilippo Syndrome or Mucopolysaccharidosis Type III B (MPS IIIB) NAGLU short-chain acyl-CoA dehydrogenase (SCAD) deficiency SCAD Scheie Syndrome (also known as Mucopolysaccharidosis Type IS (MPS IS)) IDUA, α-L-iduronidase Schizophrenia Neuregulin1 (NRG1), ERB4 (receptor for Neuregulin), Complexin1 (CPLX1), TPH1 Tryptophan hydroxylase, TPH2 Tryptophan hydroxylase 2, Neurexin 1, GSK3, GSK3a, GSK3b, 5-HTT (Slc6a4), COMT, DRD (Drd1a), SLC6A3, DAOA, DTNBP1, Dao (Dao1), TCF4,COL8A2 Secretase-related disturbances APH-1 (alpha and beta), PSEN1, NCSTN, PEN-2, Nos1, Parp1, Nat1, Nat2, CTSB, APP, APH1B, PSEN2, PSENEN, BACE1, ITM2B, CTSD, NOTCH1, TNF, INS, DYT10, ADAM17,17. APOE, ACE, STN, TP53, IL6, NGFR, IL1B, ACHE, CTNNB1, IGF1, IFNG, NRG1, CASP3, MAPK1, CDH1, APBB1, HMGCR, CREB1, PTGS2, HES1, CAT, TGFB1, ENO2, ERBB4, TRAPPC10, MAOB, NGF, MMP12, JAG1, CD40LG, PPARG, FGF2, LRP1, NOTCH4, MAPK8, PREP, NOTCH3, PRNP, CTSG, EGF, REN, CD44, SELP, GHR, ADCYAP1, INSR, GFAP, MMP3, MAPK10, SP1, MYC, CTSE, PPARA, JUN, TIMP1, IL5, IL1A, MMP9, HTR4, HSPG2, KRAS, CYCS, SMG1, IL1R1, PROK1, MAPK3, NTRK1, IL13, MME, TKT, CXCR2, CHRM1, ATXN1, PAWR, NOTCJ2, M6PR, CYP46A1, CSNK1D, MAPK14, PRG2, PRKCA, L1 CAM, CD40, NR1I2, JAG2, CTNND1, CMA1, SORT1, DLK1, THEM4, JUP, CD46, CCL11, CAV3, RNASE3, HSPA8, CASP9, CYP3A4, CCR3, TFAP2A, SCP2, CDK4, JOF1A, TCF7L2, B3GALTL, MDM2, RELA, CASP7, IDE, FANP4, CASK, ADCYAP1R1, ATF4, PDGFA, C21ORF33, SCG5, RMF123, NKFB1, ERBB2, CAV1, MMP7, TGFA,Selective IgA Deficiency Type 1: MSH5, Type 2: TNFRSF13B, RXRA, STX1A, PSMC4, P2RY2, TNFRSF21, DLG1, NUMBL, SPN, PLSCR1, UBQLN2, UBQLN1, PCSK7, SPON1, SILV, QPCT, HESS, GCC1 Petition 870250098454, dated 10 / 28 / 2025, pp. 113 / 170 106 / 141 Disease(s) Gene(s) Severe Combined Immunodeficiency (SCID), SCID-Xl and Adenosine deaminase (ADA)-SCID JAK3, JAKL, DCLRE1C, ARTEMIS, SCIDA, RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG, SCIDX1, SCIDX, IMD4, those identified in Pub. Ped. Pat. US 20110225664, 20110091441, 20100229252, 20090271881 and 20090222937 Sickle Cell Disease HBB, BCL11A, BCL11Ae, cisregulatory elements of the B-globin locus, HBG 1 / 2 promoter, distal region of the HBG CCAAT box between -92 and 130 of the HBG transcription start site, those described in WO2015148863, WO 2013 / 126794, Pub. Pat. US 20110182867 Spinocerebellar ataxias (SCA types 1, 2, 3, 6, 7, 8, 12 and 17) ATXN1, ATXN2, ATX3 Sorsby Fundus Dystrophy TIMP3 Stargardt Disease ABCR, ELOVL4, ABCA4, PROM1 Thalassemia (Alpha, Beta, Delta) HBA1, HBA2 (Alpha), HBB (Beta), HBB and HBD (delta), LCRB, BCL11A, BCL11Ae, cisregulatory elements of the β-globin locus, HBG 1 / 2 promoter, those described in WO2015148860, US Pat. Pub. 20110182867,2015 / 148860 Thymic aplasia (DiGeorge syndrome; 22q11.2 deletion syndrome) Deletion of 30 to 40 genes in the middle of chromosome 22 at a location known as 22q11.2, including TBX1, DGCR8 Transthyretin amyloidosis (ATTR) TTR (transthyretin) Trimethylaminuria FMO3 Trinucleotide repeat disorders (usually) HTT, SBMA / SMAX1 / AR, FXN / X25 ATX3, ATXN1, ATXN2, DMPK, Atrophin-1 and Atn1 (DRPLA Dx), CBP (Creb-BP - global instability), VLDLR, Atxn7, Atxn10, FEN1, TNRC6A, PABPN1, JPH3, MED15, ATXN3, TBP, CACNA1A, ATXN80S, PPP2R2B, ATXN7, TNRC6B, TNRC6C, CELF3, MAB21L1, MSH2, TMEM185A, SIX5, CNPY3, RAXE, GNB2, RPL14, ATXN8, ISR, TTR, EP400, GIGYF2, OGG1, STC1, CNDP1, C10ORF2, MAML3, DKC1, PAXIP1, CASK, MAPT, SP1, POLG, AFF2, THBS1, TP53, ESR1, CGGBP1, ABT1, KLK3, PRNP, JUN, KCNN3, BAX, FRAXA, KBTBD10, MBNL1, RAD51, NCOA3, ERDA1, TSC1, COMP, GGLC, RRAD, MSH3, DRD2, CD44, CTCF, CCND1, CLSPN, MEF2A, PTPRU, GAPDH, TRIM22, WT1, AHR, GPX1, TPMT, NDP, ARX, TYR, EGR1, UNG, NUMBL, FABP2,EN2, CRYGC, SRP14, CRYGB, PDCD1, HOXA1, ATXN2L, PMS2, GLA, CBL, FTH1, IL12RB2, OTX2, HOXA5, POLG2, DLX2, AHRR, MANF, RMEM158, see also 20110016540 Triple X Syndrome (XXX) Trisomy of chromosome X Turner Syndrome (XO) Monosomy X Tuberous Sclerosis TSC1, TSC2 Usher Syndrome (Types I, II and III) ABHD12, CDH23, CIB2, CLRN1, DFNB31, Petition 870250098454, dated 10 / 28 / 2025, pp. 114 / 170 107 / 141 Disease(s) Gene(s) GPR98, HARS, MYO7A, PCDH15, USH1C, USH1G, USH2A, USH11A, those described in WO2015134812A1 Velocardiofacial Syndrome (also known as 22q11.2 Deletion Syndrome, DiGeorge Syndrome, Conotruncal Facial Anomaly Syndrome (CTAF), Autosomal Dominant Opitz G / BB Syndrome or Cayler Cardiofacial Syndrome) Many genes are deleted, COM, TBX1 and others are associated with symptoms very long chain acyl-CoA dehydrogenase (VLCAD) deficiency VLCAD Von Gierke Disease (Glycogen Storage Disease Type I) G6PC, SLC37A4 Von Hippel-Lindau Syndrome VHL Von Hippel-Lindau Disease Willebrand (Types I, II and III) VWF Wilson's disease ATP7B Wiskott-Aldrich syndrome WAS Xeroderma pigmentosum POLH X-linked myotubular myopathy MTM1 A. Heterologous expression of a transgene for a treatment method

[0211] In some embodiments, a subject receives an effective amount of a polypeptide comprising a transgene for the expression of a protein of interest or a fragment thereof. In preferred embodiments, the polypeptide comprising a transgene is administered to the subject with an administration vehicle, such as a viral genome or an AAV vector (e.g., an rAAV vector).

[0212] In some modalities, the treatment method 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 modalities, the treatment method is intended to treat a disease or condition that leads to a defect or deficiency in multiple gene products. In some modalities, the defect or deficiency is defined by reduced expression, reduced activity, and / or aberrant localization of one or more gene products (e.g., an mRNA transcript or a protein). Petition 870250098454, dated 10 / 28 / 2025, pp. 115 / 170 108 / 141

[0213] In some modalities, the treatment method is intended to replace, supplement, or replenish a missing, 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 with a disorder or condition. In some modalities, the disorder or condition is characterized by a loss-of-function mutation or a gene deletion. In other modalities, the disorder or condition is acquired (e.g., a deficient or defective gene product of stochastic or environmental factors).

[0214] 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 of 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 with the wild-type amino acid sequence or a region therein to express a protein or fragment thereof that has enhanced biological function (e.g., increased catalytic function or reduced immunogenicity).Some examples in which a polypeptide or protein can be modified to improve biological function include mutation or addition of sites for post-translational modifications (e.g., glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation, among others, or a combination thereof), mutation or addition of cysteines for altered or added disulfide bonds, modification of binding sites for enhanced binding activity between the protein of interest and one or more known binding partners, modification of protease binding sites or targeted cleavage, modification of a signal sequence for... Petition 870250098454, dated 10 / 28 / 2025, pp. 116 / 170 109 / 141 enhanced secretion or altered localization, among other protein modifications known in the technique.

[0215] In some modalities, the treatment method is intended to increase or supplement the expression of a normally expressed protein (e.g., a protein that is present at a concentration within an accepted healthy range) in a subject with a disorder or condition that would benefit from increased expression of said protein. Increased expression of a normally expressed protein may be desired to increase the rate of an enzymatic reaction, increase the potency or rate of a signaling response (e.g., intracellularly or extracellularly), modulate the trafficking or adhesion of a cell or cellular component, increase the probability or propensity for binding or occurrence of a transient interaction (e.g., based on the affinity or Kd of two or more molecules), or otherwise modulate one or more biological processes.Such methods can be clinically useful for increasing the expression of a protein with redundant function for a deficient or defective protein. Such methods can also be clinically useful for modulating a disease-causing protein or protein fragment that is logistically more difficult to employ for a treatment method due to a variety 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.

[0216] In some modalities, the treatment method 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 modalities may be clinically useful for blocking, inhibiting, proteolyzing, mediating the clearance of, or otherwise attenuating the effect of an agent. Petition 870250098454, dated 10 / 28 / 2025, pp. 117 / 170 110 / 141 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).

[0217] In some embodiments, treatment with an effective amount of a polynucleotide comprising a transgene encoding a protein or a pharmaceutical composition containing the same increases gene expression 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 more than 100% (about 100%, about 125%, about 150%, approximately 175%, approximately 200%, approximately 225%, approximately 250%, approximately 275%, approximately 300% or more) compared to a reference, such as a biological sample from a control subject or a biological sample from the same subject before treatment administration.In some modalities, a control subject is a subject with the same disease or condition who did not receive the treatment method. In other modalities, a control subject is a healthy control subject.

[0218] In some embodiments, treatment with an effective amount of a polynucleotide comprising a transgene encoding a protein or a pharmaceutical composition containing the same increases gene expression by about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, about Petition 870250098454, dated 10 / 28 / 2025, pp. 118 / 170 111 / 141 of 20 times, approximately 25 times, approximately 30 times, approximately 35 times, approximately 40 times, approximately 45 times, approximately 50 times or more compared to a reference, such as a biological sample from a control subject or a biological sample from the same subject before treatment administration. In some modalities, a control subject is a subject with the same disease or condition who did not receive the treatment method. In other modalities, a control subject is a healthy control subject.

[0219] In some embodiments, an effective amount of a polynucleotide comprising a transgene encoding a protein or a pharmaceutical compound containing the same corresponds to a therapeutic level of gene expression. In some embodiments, a therapeutic level is a physiologically acceptable level of expression based on a healthy control subject or a median level of expression based on a plurality of healthy control subjects. In some embodiments, a therapeutic level is a level of expression that falls within a range of acceptable expression levels based on the subject’s characteristics (e.g., age, sex, weight, height, ethnicity, lifestyle, and / or one or more of the subject’s comorbidities).In other modalities, a therapeutic level is an expression level that exceeds physiological values, such that the therapeutic expression level is approximately 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, or approximately 500% higher than a physiological level. Expression based on a healthy control. Subject or a median level of expression based on a plurality of healthy control subjects.

[0220] In some modalities, an effective quantity of a Petition 870250098454, dated 10 / 28 / 2025, pp. 119 / 170 112 / 141 polynucleotide comprising a transgene encoding a protein or a pharmaceutical compound containing the same corresponds to a subtherapeutic level of gene expression. In some modalities, a subtherapeutic 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 modalities, a subtherapeutic level is an expression level that falls within a range of acceptable expression levels based on the subject's characteristics (e.g., age, sex, weight, height, ethnicity, lifestyle, and / or one or more of the subject's comorbidities).In some modalities, a subtherapeutic level is an expression level that is below a physiological value, such that the subtherapeutic expression level is approximately 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 modalities, a subtherapeutic level is a level of expression that is below a therapeutic level, such that the subtherapeutic level of expression is approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% lower than a therapeutic level.

[0221] In some embodiments, treatment with an effective amount of a polynucleotide comprising a transgene encoding a protein or a pharmaceutical compound containing the same results in ubiquitously increased gene expression, such that there is a measurable increase in all organ systems or in all tissues of the subject (e.g., throughout the body). In other embodiments, treatment with an effective amount of a polynucleotide comprising a transgene encoding a protein or a pharmaceutical compound containing the same results in locally increased gene expression, such that Petition 870250098454, dated 10 / 28 / 2025, pages 120 / 170 113 / 141 there is a measurable increase in a limited region of the subject's body (for example, in a subset of tissues, in one or more organs, or in one or more organ systems of interest).

[0222] In some modalities, the increase in gene expression after 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 for measuring mRNA transcript expression levels are routine in the field. Exemplary methods for measuring mRNA transcript expression levels include, among others, 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.

[0223] In some modalities, the increase in gene expression after 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 for measuring protein expression levels are routine in the field. Exemplary methods for measuring protein expression levels include, among others, Western blot analysis, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, flow cytometry, fluorimetry, colorimetry, spectrophotometric analyses, or an array using a linking oligonucleotide (e.g., an aptamer) or an antibody against one or more proteins of interest.

[0224] In some embodiments, increased expression of the transgene encoding a protein is maintained after administration. In some embodiments, increased expression is maintained indefinitely after administration. In some embodiments, increased expression is maintained Petition 870250098454, dated 10 / 28 / 2025, pp. 121 / 170 114 / 141 for at least one week after administration. In some modalities, increased expression is maintained between one week and 10 years after administration. In some modalities, increased expression is maintained between one week and seven years after administration. In some modalities, increased expression is maintained between one week and five years after administration. In some modalities, increased expression is maintained between one week and three years after administration. In some modalities, increased expression is maintained between one week and one year after administration.

[0225] In some modalities, increased expression is maintained between 1 week and 24 months after administration. In some modalities, increased expression is maintained between 1 week and 23 months after administration. In some modalities, increased expression is maintained between 1 week and 22 months after administration. In some modalities, increased expression is maintained between 1 week and 21 months after administration. In some modalities, increased expression is maintained between 1 week and 20 months after administration. In some modalities, increased expression is maintained between 1 week and 19 months after administration. In some modalities, increased expression is maintained between 1 week and 18 months after administration. In some modalities, increased expression is maintained between 1 week and 17 months after administration. In some modalities, increased expression is maintained between 1 week and 16 months after administration.In some modalities, increased expression is maintained between 1 week and 15 months after administration. In some modalities, increased expression is maintained between 1 week and 14 months after administration. In some modalities, increased expression is maintained between 1 week and 13 months after administration. In some modalities, increased expression is maintained between 1 week and 12 months after administration. In some... Petition 870250098454, dated 10 / 28 / 2025, pp. 122 / 170 In some modalities, increased expression is maintained between 1 week and 11 months after administration. In some modalities, increased expression is maintained between 1 week and 10 months after administration. In some modalities, increased expression is maintained between 1 week and 9 months after administration. In some modalities, increased expression is maintained between 1 week and 8 months after administration. In some modalities, increased expression is maintained between 1 week and 7 months after administration. In some modalities, increased expression is maintained between 1 week and 6 months after administration. In some modalities, increased expression is maintained between 1 week and 5 months after administration. In some modalities, increased expression is maintained between 1 week and 4 months after administration. In some modalities, increased expression is maintained between 1 week and 3 months after administration.In some modalities, increased expression is maintained between 1 week and 2 months after administration.

[0226] In some modalities, the increased expression is maintained for more than 10 years after administration. In some modalities, the increased expression is maintained between 6 months and 10 years after administration. In some modalities, the increased expression is maintained between 1 year and 10 years after administration. In some modalities, the increased expression is maintained between 2 years and 10 years after administration. In some modalities, the increased expression is maintained between 3 years and 10 years after administration. In some modalities, the increased expression is maintained between 4 years and 10 years after administration. In some modalities, the increased expression is maintained between 4 years and 10 years after administration. In some modalities, the increased expression is maintained between 5 years and 10 years after administration. In some modalities, the Petition 870250098454, dated 10 / 28 / 2025, pp. 123 / 170 116 / 141 Increased expression is maintained between 6 and 10 years after administration. In some modalities, increased expression is maintained between 7 and 10 years after administration. In some modalities, increased expression is maintained between 8 and 10 years after administration. In some modalities, increased expression is maintained between 9 and 10 years after administration.

[0227] The magnitude or duration of increased expression of the transgene-encoded mRNA and / or protein transcript after administration of the polynucleotide or pharmaceutical composition comprising it may depend on one or more factors. Such factors may include, but are not limited to, the route of administration, the dose, the stability of the mRNA and / or protein transcript, or the disease or condition for which the treatment is intended, and / or one or more comorbidities of the subject.

[0228] As described in the previous section, in some embodiments, the magnitude or duration of increased expression of the transgene-encoded mRNA transcript and / or protein is further modulated by the administration of one or more inhibitory nucleic acid molecules (e.g., one or more inhibitory RNA molecules).

[0229] In some embodiments, the treatment method includes administering an effective amount of a polynucleotide comprising a transgene or a pharmaceutical composition containing the same to the subject (e.g., a human) by means of in vivo, in vitro, or ex vivo administration methods, or any combination thereof. In some embodiments, the treatment method includes in vivo administration methods, such that the polynucleotide or pharmaceutical composition is administered directly to the subject's body by an appropriate route of administration, such as one or more of the methods described below. In some embodiments, the treatment method includes in vitro or ex vivo administration methods, such that the polynucleotide or composition Petition 870250098454, dated 10 / 28 / 2025, pp. 124 / 170 117 / 141 pharmaceutical containing the same is administered to one or more isolated cells or tissues of the subject and then, optionally, implanted, dispensed or deposited separately in the subject's body.

[0230] The treatment method involves administering an effective amount of a polynucleotide comprising a transgene for the expression of a protein of interest or a fragment thereof or a pharmaceutical composition containing 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, subcutaneous, intradermal, intravenous, intraperitoneal, intra-arterial, intraventricular, intranasal, intraorbital, intracranial, or intraosseous administration. In some embodiments, the treatment method involves administering a codon-optimized gene product or a pharmaceutical composition containing the same to a subject to produce a therapeutic effect.

[0231] In some embodiments, an effective amount of a polypeptide comprising a transgene or a pharmaceutical composition containing 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 composition containing the same is administered to a subject between 1 and 5 times. In other embodiments, an effective amount of a polypeptide comprising a transgene or a pharmaceutical composition containing the same is administered to a subject between 3 and 5 times. In other embodiments, an effective amount of a polypeptide comprising a transgene or a pharmaceutical composition containing the same is administered to a subject between 1 and 3 times. B. Reduction of Heterologous Transgene Expression for a Treatment Method

[0232] After administering an effective amount of one or Petition 870250098454, dated 10 / 28 / 2025, pp. 125 / 170 118 / 141 more polynucleotides comprising a transgene or one or more pharmaceutical compositions containing the same, it may be desirable to reduce the expression of the transgene. Reducing expression may refer to dampening, decreasing, or minimizing the potency of the polynucleotide comprising the transgene. For example, a subject who has received a polynucleotide comprising a transgene or a pharmaceutical composition containing the same may have a contraindication for which it is desirable to temporarily reduce the expression of the transgene. Reducing transgene expression may be useful or recommended to a subject by a qualified professional (e.g., a physician or clinician) for potential contraindications such as an acute infection, a surgical procedure, treatment with cytotoxic or chemotherapeutic agents, or fertility and / or pregnancy treatments of the subject.

[0233] The reduction of the expression of a polynucleotide comprising a transgene or a pharmaceutical composition containing it may be achieved by administering one or more inhibitory nucleic acids (e.g., one or more inhibitory nucleic acids; for example, one or more unmodified or modified inhibitory nucleic acids) or one or more pharmaceutical compositions containing it, as described herein.

[0234] One or more inhibitory nucleic acids or pharmaceutical compositions containing them may be administered to a subject to reduce the expression of the polynucleotide comprising the transgene temporarily or transiently. In some embodiments, an inhibitory nucleic acid or a pharmaceutical composition containing it is administered as a single administration. In some embodiments, an inhibitory nucleic acid or a pharmaceutical composition containing it is administered as a plurality of administrations.

[0235] An inhibitory nucleic acid can be administered to a Petition 870250098454, dated 10 / 28 / 2025, pp. 126 / 170 119 / 141 subject to any time after administration of a polynucleotide comprising a transgene. In some embodiments, the inhibitory nucleic acid is administered at least one week after administration of the polynucleotide. In some embodiments, the inhibitory nucleic acid is administered between one week and 10 years after administration of the polynucleotide. In some embodiments, the inhibitory nucleic acid is administered between one week and seven years after administration of the polynucleotide. In some embodiments, the inhibitory nucleic acid is administered between one week and five years after administration of the polynucleotide. In some embodiments, the inhibitory nucleic acid is administered between one week and three years after administration of the polynucleotide. In some embodiments, the inhibitory nucleic acid is administered between one week and one year after administration of the polynucleotide.

[0236] In some embodiments, the inhibitory nucleic acid is administered between 1 week and 24 months after polynucleotide administration. In some embodiments, the inhibitory nucleic acid is administered between 1 week and 23 months after polynucleotide administration. In some embodiments, the inhibitory nucleic acid is administered between 1 week and 22 months after polynucleotide administration. In some embodiments, the inhibitory nucleic acid is administered between 1 week and 21 months after polynucleotide administration. In some embodiments, the inhibitory nucleic acid is administered between 1 week and 20 months after polynucleotide administration. In some embodiments, the inhibitory nucleic acid is administered between 1 week and 19 months after polynucleotide administration. In some embodiments, the inhibitory nucleic acid is administered between 1 week and 18 months after polynucleotide administration. In some embodiments, the inhibitory nucleic acid is Petition 870250098454, dated 10 / 28 / 2025, pp. 127 / 170 120 / 141 administered between polynucleotides. In administered between polynucleotides. In administered between polynucleotides. In administered between polynucleotides. In administered between polynucleotides. In administered between polynucleotides. In administered between polynucleotides. In administered between polynucleotides. In administered between polynucleotides. In administered between polynucleotides. In administered between polynucleotides. In administered between polynucleotides. In administered between polynucleotides. In administered between polynucleotides. In administered between polynucleotides. In some modalities, one week and 17 months; in some modalities, one week and 16 months; in some modalities, one week and 15 months; in some modalities, one week and 14 months; in some modalities, one week and 13 months; in some modalities, one week and 12 months; in some modalities, one week and 11 months; in some modalities, one week and 10 months; in some modalities, one week and 9 months; in some modalities, one week and 8 months; in some modalities, one week and 7 months.1 week and 6 months in some modalities, 1 week and 5 months in some modalities, 1 week and 4 months in some modalities, after administration of the inhibitory nucleic acid is after administration of the inhibitory nucleic acid is after administration of the inhibitory nucleic acid is after administration of the inhibitory nucleic acid is after administration of the inhibitory nucleic acid is after administration of the inhibitory nucleic acid is after administration of the inhibitory nucleic acid is after administration of the inhibitory nucleic acid is after administration of the inhibitory nucleic acid is after administration of the inhibitory nucleic acid is after administration of the inhibitory nucleic acid is after administration of the inhibitory nucleic acid is after administration of the inhibitory nucleic acid is after administration of the inhibitory nucleic acid is after administration of the inhibitory nucleic acid is, Petition 870250098454, dated 10 / 28 / 2025, pages 128 / 170 121 / 141 administered between 1 week and 3 months after polynucleotide administration. In some embodiments, the inhibitory nucleic acid is administered between 1 week and 2 months after polynucleotide administration. In some embodiments, the inhibitory nucleic acid is administered between 1 week and 1 month after polynucleotide administration.

[0237] In some embodiments, treatment with an effective amount of an inhibitory nucleic acid or a pharmaceutical composition containing the same reduces gene expression (e.g., transgene) 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 in some cases, more than 100%, compared to a reference, such as a biological sample from a subject of A control subject is a biological sample from the same subject before treatment administration. In some modalities, a control subject is a subject with the same disease or condition who did not receive the treatment method. In other modalities, a control subject is a healthy control subject.

[0238] In some embodiments, treatment with an effective amount of an inhibitory nucleic acid or a pharmaceutical composition containing the same reduces gene expression (e.g., transgene) by about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, Petition 870250098454, dated 10 / 28 / 2025, pp. 129 / 170 122 / 141 in approximately 18 times, approximately 19 times, approximately 20 times, approximately 25 times, approximately 30 times, approximately 35 times, approximately 40 times, approximately 45 times, approximately 50 times or more compared to a reference, such as a biological sample from a control subject or a biological sample from the same subject before treatment administration. In some modalities, a control subject is a subject with the same disease or condition who did not receive the treatment method. In other modalities, a control subject is a healthy control subject.

[0239] In some embodiments, an effective amount of an inhibitory nucleic acid or a pharmaceutical composition containing the same corresponds to a therapeutic level of gene expression. In some embodiments, a therapeutic level is an expression level that is lower than the gene expression level after administration of a polynucleotide comprising a transgene, such that the therapeutic expression level is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100% or more than 100%, as applicable, relative to the gene expression level after administration of the polynucleotide comprising a transgene. In some modalities, a therapeutic level is a physiologically acceptable level of expression based on a healthy control subject or a median level of expression based on a plurality of healthy control subjects.In some modalities, a therapeutic level is a level of expression that falls within a range of accepted levels of expression based on the subject's characteristics (e.g., age, sex, weight, height, ethnicity, lifestyle, and / or one or more comorbidities of the subject).

[0240] In some embodiments, an effective amount of an inhibitory nucleic acid or a pharmaceutical composition containing it corresponds to a subtherapeutic level of gene expression. In some Petition 870250098454, dated 10 / 28 / 2025, pp. 130 / 170 In 123 / 141 modalities, a subtherapeutic level is a level of expression that is lower than the therapeutic level, such that the subtherapeutic level of expression is approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% lower than a therapeutic level. In some modalities, a subtherapeutic level is a level of expression that falls within a range of accepted levels of expression based on the subject's characteristics (e.g., age, sex, weight, height, ethnicity, lifestyle, and / or one or more comorbidities of the subject). In some modalities, a therapeutic level is a physiologically acceptable level of expression based on a healthy control subject or a median level of expression based on a plurality of healthy control subjects.In some modalities, a subtherapeutic level is an expression level that is below a physiological value, such that the subtherapeutic expression level is approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% lower than a physiological value.

[0241] In other embodiments, an effective amount of an inhibitory molecule or of a pharmaceutical composition containing the same results in a gene expression level that is comparable to a gene expression level measured in a sample obtained from the subject before administration of the polynucleotide comprising the transgene (i.e., levels prior to heterologous expression of the transgene).

[0242] In some embodiments, treatment with an effective amount of an inhibitory nucleic acid or a pharmaceutical composition containing the same results in ubiquitously reduced expression of the gene (e.g., the transgene), such that there is a measurable reduction in the gene in all organ systems or in all tissues of the subject (e.g., throughout the body). In other embodiments, treatment with an effective amount of an inhibitory nucleic acid or a composition Petition 870250098454, dated 10 / 28 / 2025, pp. 131 / 170 124 / 141 pharmaceutical containing the same results in locally reduced expression of the gene (e.g., the transgene), such that there is a measurable decrease in a limited region of the body (e.g., in a subset of tissues, in one or more organs, or in one or more organ systems of interest).

[0243] In some modalities, decreased transgene expression after treatment is measured by a decrease in mRNA transcript levels or concentrations (e.g., an mRNA transcript that corresponds to the transgene) relative to a reference. Methods for measuring mRNA transcript expression levels are routine in the field. Exemplary methods for measuring mRNA transcript expression levels include, among others, 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.

[0244] In some modalities, reduced transgene expression after treatment is measured by a reduction in protein expression levels or concentrations (e.g., a protein encoded by the transgene) relative to a reference. Methods for measuring protein expression levels are routine in the field. Exemplary methods for measuring protein expression levels include, among others, Western blot analysis, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, flow cytometry, fluorimetry, colorimetry, spectrophotometric analyses, or an array using a linking oligonucleotide (e.g., an aptamer) or an antibody against one or more proteins of interest.

[0245] In some embodiments, the reduced expression of the transgene encoding a protein is maintained for a certain period of Petition 870250098454, dated 10 / 28 / 2025, pp. 132 / 170 125 / 141 time after administration. In some modalities, the reduced expression is maintained for at least one week after administration. In some modalities, the reduced expression is maintained between one week and 10 years after administration. In some modalities, the reduced expression is maintained between one week and nine years after administration. In some modalities, the reduced expression is maintained between one week and eight years after administration. In some modalities, the reduced expression is maintained between one week and seven years after administration. In some modalities, the reduced expression is maintained between one week and six years after administration. In some modalities, the reduced expression is maintained between one week and five years after administration. In some modalities, the reduced expression is maintained between one week and four years after administration. In some modalities, the reduced expression is maintained between one week and three years after administration.In some modalities, reduced expression is maintained between 1 week and 2 years after administration.

[0246] In some modalities, the reduced expression is maintained between 1 week and 24 months after administration. In some modalities, the reduced expression is maintained between 1 week and 23 months after administration. In some modalities, the reduced expression is maintained between 1 week and 22 months after administration. In some modalities, the reduced expression is maintained between 1 week and 21 months after administration. In some modalities, the reduced expression is maintained between 1 week and 20 months after administration. In some modalities, the reduced expression is maintained between 1 week and 19 months after administration. In some modalities, the reduced expression is maintained between 1 week and 18 months after administration. In some modalities, the reduced expression is maintained between 1 week and 17 months after administration. In some modalities, the reduced expression Petition 870250098454, dated 10 / 28 / 2025, pp. 133 / 170 The reduced expression 126 / 141 is maintained between 1 week and 16 months after administration. In some modalities, the reduced expression is maintained between 1 week and 15 months after administration. In some modalities, the reduced expression is maintained between 1 week and 14 months after administration. In some modalities, the reduced expression is maintained between 1 week and 13 months after administration. In some modalities, the reduced expression is maintained between 1 week and 12 months after administration. In some modalities, the reduced expression is maintained between 1 week and 11 months after administration. In some modalities, the reduced expression is maintained between 1 week and 10 months after administration. In some modalities, the reduced expression is maintained between 1 week and 9 months after administration. In some modalities, the reduced expression is maintained between 1 week and 8 months after administration. In some modalities, the reduced expression is maintained between 1 week and 7 months after administration.In some modalities, the reduced expression is maintained between 1 week and 6 months after administration. In some modalities, the reduced expression is maintained between 1 week and 5 months after administration. In some modalities, the reduced expression is maintained between 1 week and 4 months after administration. In some modalities, the reduced expression is maintained between 1 week and 3 months after administration. In some modalities, the reduced expression is maintained between 1 week and 2 months after administration. In some modalities, the reduced expression is maintained between 1 week and 1 month after administration.

[0247] In some modalities, the abbreviated expression is maintained for approximately 5 years after administration. In some modalities, the abbreviated expression is maintained for between 6 months and 5 years after administration. In some modalities, the abbreviated expression is maintained for between 1 year and 5 years after administration. In some modalities, the abbreviated expression is maintained for between 2 years and 5 years after administration. In some modalities, Petition 870250098454, dated 10 / 28 / 2025, pp. 134 / 170 127 / 141 the reduced expression is maintained between 3 and 5 years after administration. In some modalities, the reduced expression is maintained between 4 and 5 years after administration.

[0248] The magnitude or duration of reduced expression of the transgene-encoded mRNA transcript and / or protein after administration of the inhibitory nucleic acid or pharmaceutical composition containing it may depend on one or more factors. Such factors may include, but are not limited to, the route of administration, the dose, the 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.

[0249] In some embodiments, the treatment method includes administering an effective amount of a polynucleotide comprising a transgene for the expression of a protein of interest or a fragment thereof or a pharmaceutical composition containing it by means of in vivo, in vitro, or ex vivo administration methods or any combination thereof. In some embodiments, the treatment method includes in vivo administration methods, whereby the polynucleotide or pharmaceutical composition is administered directly to the subject’s body. In some embodiments, the treatment method includes in vitro or ex vivo administration methods, whereby the polynucleotide or pharmaceutical composition containing the same is administered to one or more isolated cells or tissues of the subject and then implanted, dispensed, or deposited separately in the subject’s body.

[0250] The treatment method involves administering an effective amount of an inhibitory nucleic acid or a pharmaceutical composition containing the same to the subject (e.g., a human being) by any appropriate route of administration. Appropriate routes of administration include, but are not limited to, intramuscular, subcutaneous, intradermal, intravenous, intraperitoneal, intra-arterial, intraventricular, intranasal, intraorbital administration. Petition 870250098454, dated 10 / 28 / 2025, pages 135 / 170 128 / 141 intracranial or intraosseous. In some modalities, the treatment method involves administering a codon-optimized gene product or a pharmaceutical composition containing the same to a subject to produce a therapeutic effect.

[0251] In preferred embodiments, the route of administration of a polynucleotide comprising the transgene and an inhibitory nucleic acid or pharmaceutical compositions thereof are the same. In other embodiments, a polynucleotide comprising a transgene and an inhibitory nucleic acid or pharmaceutical compositions thereof are administered within a certain distance of a body region of a subject who needs it (e.g., a human being). In some embodiments, a polynucleotide comprising a transgene and an inhibitory nucleic acid or pharmaceutical compositions thereof are administered at a distance of 10 cm or closer (e.g., at a distance of 10 cm, at a distance of 9 cm, at a distance of 8 cm, at a distance of 7 cm, at a distance of 6 cm, at a distance of 5 cm, at a distance of 4 cm, at a distance of 3 cm, at a distance of 2 cm, at a distance of 1 cm).In some embodiments, a polynucleotide comprising a transgene and an inhibitory nucleic acid or pharmaceutical compositions thereof are administered at a distance of 3 cm or closer (e.g., at a distance of 3 cm, at a distance of 2 cm, at a distance of 1 cm). In other embodiments, a polynucleotide comprising a transgene and an inhibitory nucleic acid 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 on the subject's body for administration of a polynucleotide comprising a transgene and an inhibitory nucleic acid or pharmaceutical compositions thereof is... Petition 870250098454, dated 10 / 28 / 2025, pp. 136 / 170 129 / 141 marked before administration, for example, by a medical tattoo. V. Kits

[0252] The compositions or methods described herein may be provided in a kit for use in the expression of a transgene and / or subsequent reduction of expression. In some embodiments, the compositions and methods described herein may be provided in a kit for use in the treatment of a disease or condition. In some embodiments, the kit may include a package insert instructing the kit user to construct or synthesize a polynucleotide comprising a transgene, a vector comprising the same, an inhibitory nucleic acid, or a pharmaceutical composition described herein. In other embodiments, the kit may include a package insert instructing the kit user to perform any of the treatment methods described herein. The kit may optionally include a syringe or device for administering the compositions of this 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 protein of interest. EXAMPLES Example 1: Intramuscular administration of an siRNA reduces the expression of a transgene delivered with an AAV vector.

[0253] This example describes the design and intramuscular delivery of siRNA molecules to reduce the expression of a transgene of interest after delivery of a transgene with a designed AAV vector. An AAV template genome in an AAV9 serotype was designed to have multiple potential siRNA target sites in the 3'UTR of a transgene (FIG. 1), in which the potential siRNA target sites had no sequence identity with a region in a host genome (e.g., a Petition 870250098454, dated 10 / 28 / 2025, pp. 137 / 170 130 / 141 mouse genome; for example, a human genome). The transgene (e.g., the therapeutic transgene) was cyno dulaglutide (CyDula), a GLP-1 agonist protein that resembles dulaglutide but is modified to reduce immunogenicity in cynomolgus monkeys.

[0254] The siRNA target region in the transgene contained 7 distinct and non-overlapping siRNA target sequences. For the proof-of-concept experiment, a single siRNA target sequence was chosen. Three siRNAs targeting the chosen siRNA target site in the 3'UTR transgene were synthesized with different phosphorothioate linkages, 2'-O-methyl (2'OMe) modifications, and 2'-fluoro modifications. In addition, two of these siRNAs also contained a hydrophobic moiety. Besides these experimental siRNA molecules, three negative control scrambled sequence siRNA molecules were synthesized to match the chemical modification patterns of the designed siRNA, and sequencing alignment data confirmed that these siRNA molecules did not share complementarity with any site in the mouse genome.These negative control siRNA molecules would allow the detection of any adverse impact of transgenic expression from intramuscular administration of an siRNA in the absence of a specific gene silencing effect.

[0255] Following the design of the AAV genome and siRNA synthesis, seven groups of cloth mice (8 mice per group) were injected into the hind leg muscle with 2 x 109 copies of the genome (GC) of the AAV9-CyDula construct. Thirteen days after administration of the AAV vector, a blood sample was obtained from each mouse to establish a baseline of CyDula expression. The following day, two weeks after injection of the AAV construct, each mouse was injected with either a suitable siRNA molecule or a control. As negative controls, one group of mice was injected with phosphate-buffered saline (PBS) and Petition 870250098454, dated 10 / 28 / 2025, pages 138 / 170 131 / 141 another group did not receive any injection (untreated control). Two weeks after siRNA administration, and every two weeks thereafter, blood samples were obtained from the mice to measure potential changes in serum CyDula expression levels via ELISA (FIG. 2).

[0256] Untreated mice, as well as mice administered PBS or negative control scrambled siRNA sequences (“C1” and “C1-HM”), where C1 is an unmodified scrambled siRNA and C1-HM is a scrambled siRNA that is modified to contain a hydrophobic moiety. All three groups showed no changes in CyDula protein expression during a 3-month period after PBS or siRNA administration (FIGS. 3A-B). These results confirm that injection of a non-hybridizing nucleotide siRNA does not affect the expression of an intramuscularly administered AAV transgene.

[0257] Mice treated with 200 μg of a T1-targeted siRNA did not show significant changes in CyDula protein expression over time, similar to untreated and PBS-treated controls. On the other hand, mice treated with 200 μg of an equivalent targeted siRNA containing a hydrophobic fraction modification in the sense strand (T1-HM) showed a significant and lasting reduction in CyDula protein expression. Fourteen days after T1-HM administration, CyDula protein expression was reduced by approximately 65% ​​compared to baseline levels obtained before siRNA administration (FIG. 4). These results indicate that a cholesterol modification significantly increases the cellular uptake of an siRNA molecule after intramuscular administration.

[0258] Next, the durability of reduced transgene expression was evaluated by intramuscular administration of cholesterol-modified siRNA molecules. Approximately three months after receiving 200 μg Petition 870250098454, dated 10 / 28 / 2025, pp. 139 / 170 132 / 141 of the hydrophobic T1-HM-modified targeting siRNA, mice recovered only about 40% of the lost maximum expression (FIG. 5). Furthermore, administration of 50 μg of a similar siRNA produced a smaller magnitude of reduction in CyDula protein expression compared to the reduction achieved with 200 μg; however, similar to the higher dose, this reduction was equally long-lasting, with mice recovering about 50% of the lost maximum expression by day 122 of the study (FIG. 6). These data collectively show that administration of a hydrophobic-modified siRNA that targets and hybridizes to a specific region of a transgene results in a surprisingly long-lasting and durable reduction in transgene expression. Example 2: Response of serum reporter expression delivered by IMAAV to administration of multiple doses of siRNA targeting the 3'UTR of the AAV transgene.

[0259] This example describes the design and intramuscular delivery of siRNA molecules to reduce the expression of a transgene of interest after administration of a transgene with a designed AAV vector. An AAV template genome in an AAV9 serotype was designed to have multiple potential siRNA target sites in the 3'UTR of a transgene (FIG. 1), wherein the potential siRNA target sites had no sequence identity with a region in a host genome (e.g., a mouse genome; e.g., a human genome). The transgene (e.g., the therapeutic transgene) was cyno dulaglutide (CyDula), a GLP-1 agonist protein that resembles dulaglutide but is modified to reduce immunogenicity in cynomolgus monkeys.

[0260] An experimental siRNA (siRNA-1) was synthesized to target a sequence in the 3'UTR of the AAV transgene. The experimental siRNA contained a hydrophobic moiety modification at the 3' end to Petition 870250098454, dated 10 / 28 / 2025, pages 140 / 170 133 / 141 increase muscle uptake, as well as 2'O-Me and 2'-fluoro modifications. In addition to the experimental siRNA molecule, a scrambled sequence negative control siRNA molecule ('non-targeted control oligo') was synthesized to match the chemical modification patterns of the designed siRNA. Sequencing alignment data confirmed that the siRNA molecules did not share complementarity with any site in the mouse genome. The negative control siRNA molecule would allow the detection of any adverse impact of transgenic expression from intramuscular administration of an siRNA in the absence of a gene-specific silencing effect.

[0261] After AAV genome design and siRNA synthesis, 13 groups of cloth mice (8 mice per group) were injected into the hind leg muscle with 2 x 109 copies of the AAV9-CyDula construct genome (GC). Thirteen days after AAV vector administration, a blood sample was obtained from each mouse to establish a baseline of CyDula expression. The following day, two weeks after injection of the AAV construct, each mouse was injected with either a siRNA molecule (400 μg, 200 μg, 100 μg, 50 μg, 25 μg, 12 μg, or 6 μg per mouse in different groups) or the non-targeted control oligo (400 μg, 200 μg, 100 μg, 50 μg per mouse in different groups) at varying doses to establish the relationship between siRNA dose and the reduction in serum reporter transgene expression. As negative controls, one group of mice was injected with PBS.Two weeks after siRNA administration, blood samples were obtained from the mice to measure potential changes in serum CyDula expression levels via ELISA.

[0262] We compared the mean fraction of the CyDula (GLP-1Fc) group remaining in serum on d28 (2 weeks after siRNA treatment) compared to the expression level on d13, before siRNA, with a single siRNA. Petition 870250098454, dated 10 / 28 / 2025, pp. 141 / 170 134 / 141 scrambled as a negative control (FIG. 7). The PBS-treated group did not show a significant decrease in serum CyDula expression. Mice treated with at least 50 μg of siRNA-1 showed dose-sensitive reductions in serum CyDula, with the highest dose of 400 μg / mouse achieving a >60% reduction in reporter expression. In contrast, the untargeted siRNA did not lead to a reduction in reporter expression. These data demonstrate that administration of a cholesterol-modified siRNA that targets and hybridizes to a specific region of a transgene results in a dose-sensitive reduction in serum transgene expression. Example 3: Extent of transgene expression reduction in muscle after administration of multiple doses of siRNA targeting the 3'UTR of the AAV transgene.

[0263] The murine model for IM-AAV differs from larger animal models in that a significant portion of the injected AAV vector spreads to the liver in the murine model, but much less so in larger animals such as non-human primates and presumably humans. This confounds efforts to understand the extent of transgene expression reduction in muscle using the murine model, since siRNAs injected into muscle can reach muscle much more efficiently than the liver. Liver expression may therefore contribute a background level of serum reporter protein that obscures the true extent of muscle transgene expression loss. Because muscle transgene expression loss is the critical parameter in large animal (and human) systems, we conducted an experiment designed to specifically measure muscle transgene expression reduction.

[0264] Two experimental siRNAs were synthesized (siRNA-1 and siRNA-2). siRNA-1 and siRNA-2 target the same sequence within the 3'UTR of the AAV-CyDula genome (FIG. 1). All experimental siRNAs Petition 870250098454, dated 10 / 28 / 2025, pp. 142 / 170 135 / 141 contained a hydrophobic moiety modification at the 3' end to increase muscle uptake, as well as 2'O-Me and 2'-fluoro modifications. siRNA-1 and siRNA-2 differ slightly in design, including differences in the pattern of the 2'O-Me and 2'-fluoro modifications.

[0265] Thirteen groups of cloth mice (8 mice per group) were injected into the hind leg muscle with 2 x 109 copies of the genome (GC) of the AAV9-CyDula construct. Thirteen days after AAV vector administration, a blood sample was obtained from each mouse to establish a baseline of CyDula expression. The following day, two weeks after AAV construct injection, each mouse was injected with a specific dose of one of the siRNA molecules. As a negative control, one group of mice was injected with PBS. Two weeks after siRNA administration, blood samples were obtained from the mice to measure potential changes in serum CyDula expression levels via ELISA, and the mice were sacrificed with the injected muscle tissue and a liver sample obtained for transgene mRNA analysis.

[0266] Administration of siRNA-1 and siRNA-2 resulted in reduced serum CyDula levels compared to a PBS-administered control at a 2-week time point after post-IM-siRNA administration (FIG. 8). As expected, no reduction in serum CyDula protein levels was observed for PBS. siRNA-1 achieved a significant dose-sensitive reduction in serum CyDula, as previously observed. siRNA-2 achieved a slightly higher level of CyDula reduction (almost 80% at the 200 μg dose), indicating that specific siRNA design parameters may result in different initial efficacies of siRNAs targeting the same vector sequence.

[0267] Administration of siRNA-1 or siRNA-2 also resulted in reduced levels of transgene mRNA in muscle tissue compared to a Petition 870250098454, dated 10 / 28 / 2025, pp. 143 / 170 136 / 141 control administered with PBS, as measured by qPCR using a transgene-specific primer and probe set (FIG. 9). These data confirm the greater potency of siRNA-2 compared to siRNA-1 observed in serum data (FIG. 8). When measured at the level of muscle transgene expression (transgenic muscle mRNA), siRNA-1 and siRNA-2 show a more extensive reduction than serum protein measurements would suggest (~92% reduction in muscle mRNA for siRNA-2 at the 200 μg dose vs. 80% reduction in serum protein expression). This may be the result of differential efficiencies of siRNA reduction in separate groups of transduced muscle and liver cells that likely contribute to serum expression of the transgenic protein.However, these results confirm an excellent local reduction in muscle of up to 92%, a level that would be clinically significant for a temporary pause in gene therapy, as this would, in many cases, remove the transgene from a therapeutic window of expression.

[0268] To better explore the potency of experimental siRNA-1, the remaining groups in the study received a series of decreasing doses of siRNA starting from a maximum dose of 400 μg / mouse. Comparing serum CyDula expression 2 weeks after siRNA administration with pre-siRNA serum CyDula levels, we observed the expected dose-response for siRNA-1, here fitting a simple three-parameter inhibition model to an IC50 of 39 μg with a maximum reduction of 30% (FIG. 10). As discussed above, liver contributions may mask the true extent of local muscle reduction in the murine model. Therefore, we evaluated the same cohort of siRNA-1-dosed groups for transgene mRNA levels in injected muscle using qPCR and transgene-specific primers and probes. Figure 11 shows the dose-response curve for siRNA-1 on injected muscle transgene mRNA levels (relative to PBS control). In terms of muscle mRNA, the Petition 870250098454, dated 10 / 28 / 2025, pages 144 / 170 137 / 141 siRNA-1 potency is slightly higher (28 μg) with a slightly lower maximum inhibition level (<10% residual expression), consistent with the higher activity of locally administered intramuscular siRNAs in injected muscle tissue—a critical parameter for larger animal studies. Example 4: Administration of an inhibitory nucleic acid successfully interrupts the expression of a heterologously expressed protein.

[0269] This example illustrates that an inhibitory nucleic acid administered intramuscularly can decrease the expression of a transgene of interest for a subject in whom the pause in expression has a clinical benefit.

[0270] A woman with a hereditary bleeding disorder has previously undergone gene therapy to increase the expression of a circulating blood clotting protein to reduce the risk of bleeding. The gene therapy included the administration of a recombinant AAV vector with the transgene of interest. After administration of the AAV comprising the transgene of interest, the relative protein expression levels detected in a blood sample from the subject are determined to be within a normal range (e.g., an expression level within an accepted range). After administration of the transgene (e.g., one or more days, one or more weeks, one or more months, or one or more years after administration of the transgene), it is determined that the subject is pregnant.

[0271] A qualified professional (e.g., a clinician) determines that the level of circulating protein expression has increased relative to pre-pregnancy expression levels and represents a potential risk for an adverse event (e.g., a blood clot and / or preeclampsia). To reduce the risk of developing one or more adverse events, the subject undergoes treatment with an inhibitory nucleic acid (e.g., Petition 870250098454, dated 10 / 28 / 2025, pages 145 / 170 138 / 141 an siRNA; for example, an siRNA modified to contain a hydrophobic moiety) to reduce the expression of the transgene encoding the circulating protein. The inhibitory nucleic acid is complementary to a region of the transcript transcribed by the transgene (for example, a region upstream of the start codon, a region downstream of the stop codon, a region in the 5'UTR, a region in the 3'UTR, or another suitable region described herein), but is not complementary to a region in the subject's genome (for example, a DNA sequence or an RNA sequence; for example, an endogenous mRNA sequence encoding the protein of interest). Thus, administration of the inhibitory nucleic acid will reduce the protein expressed by the transgene, but will not affect the endogenous protein expression of the blood clotting protein.The subject receives an effective amount of the inhibitory nucleic acid via intramuscular injection, sufficient to reduce transgene expression for the remainder of the subject's pregnancy (e.g., approximately 9 months, approximately 8 months, approximately 7 months, approximately 6 months, approximately 5 months, approximately 4 months, approximately 3 months, approximately 2 months, or approximately 1 month). Measurements of protein expression levels in a blood sample obtained from the subject determine that protein expression levels are within the normal range. Example 5: Temporal modulation of transgene expression for effective treatment of a disease or condition.

[0272] This example illustrates a method of temporal modulation of the expression of a gene, such as a transgene encoding a polypeptide or a protein of interest or a fragment thereof (e.g., a therapeutic polypeptide or a therapeutic protein or fragment thereof), by administering to a subject a polynucleotide comprising the transgene to increase the expression of the transgene and then administering to the subject an inhibitory nucleic acid to decrease the expression of the transgene.

[0273] A subject (for example, a human subject) has or is Petition 870250098454, dated 10 / 28 / 2025, pages 146 / 170 139 / 141 at risk of having a disease or condition (for example, a disease or condition listed in Table 1) is treated with a form of gene therapy comprising administering intramuscularly to the subject an effective amount of a pharmaceutical composition comprising a polynucleotide comprising (i) a transgene encoding a therapeutic polypeptide or a therapeutic protein or a fragment thereof and (ii) a region having complementarity with one or more inhibitory nucleic acids (for example, an inhibitory RNA molecule). The pharmaceutical composition may further comprise a delivery vehicle for effective delivery of the polynucleotide to the subject, wherein the delivery vehicle is a vector, such as a viral genome (for example, a recombinant AAV vector), a liposome or a microvesicle, among other suitable delivery forms described herein.The pharmaceutical composition may also include components of a genetic engineering system (e.g., CRISPR or a transposon system) to deliver the polynucleotide to the subject.

[0274] After intramuscular administration of the pharmaceutical composition comprising the polynucleotide (e.g., more than one day after, more than one week after, more than one month after, more than one year after and / or more than five years after), a biological sample (e.g., a blood sample or a biopsy sample) is obtained from the subject and confirms that the measured expression levels of the transgene (e.g., protein expression levels and mRNA transcript expression levels) are comparable to the expression levels detected in an appropriate control (e.g., a biological sample from a healthy subject (e.g., a subject who does not have or is not at risk of having the subject's disease or condition) and / or an accepted range of expression levels).

[0275] After administering the pharmaceutical composition comprising the polynucleotide (for example, more than one day later, more than Petition 870250098454, dated 10 / 28 / 2025, pages 147 / 170 140 / 141 one week later, more than one month later, more than one year later and / or more than five years later) and, optionally, determine an increase in transgene expression, the subject is determined to have or be at risk of having an indication for which a subsequent decrease in transgene expression is desirable. The subject also receives intramuscularly an effective amount of a pharmaceutical composition comprising an inhibitory nucleic acid (e.g., an inhibitory RNA molecule; e.g., an siRNA, a dsRNA, a miRNA, a shRNA, an ASO, a gapmer), wherein the inhibitory nucleic acid may further comprise one or more modifications, such as one or more modifications described herein.The inhibitory nucleic acid comprises a region that is complementary (at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% complementary) to a region of the transgene (e.g., an mRNA transcript transcribed by the transgene). Administration of the pharmaceutical composition comprising an inhibitory nucleic acid reduces the level of transgene expression by approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, or approximately 100% compared to the level of transgene expression before administration of the pharmaceutical composition comprising an inhibitory nucleic acid, indicating that the treatment to decrease transgene expression is successful. Other Modalities

[0276] All publications, patents and patent applications mentioned in this descriptive report are incorporated herein by reference to the same extent as if each independent publication or patent application were specifically and individually indicated for incorporation. Petition 870250098454, dated 10 / 28 / 2025, pages 148 / 170 141 / 141 per reference.

[0277] Although the invention has been described in connection with specific embodiments thereof, it will be understood that it is also susceptible to further modifications, and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that fall within the known or customary practice of the art to which the invention belongs and that can be applied to the essential features set forth above in this document, and follows within the scope of the claims.

[0278] Other modalities are within the scope of the claims. Petition 870250098454, dated 10 / 28 / 2025, pp. 149 / 170

Claims

1 / 8 CLAIMS 1. METHOD FOR REDUCING EXPRESSION OR ACTIVITY OF A PROTEIN of interest in a subject, the method characterized by comprising: (a) administering to the subject a recombinant adeno-associated viral vector (AAV) comprising (i) a transgene encoding the protein of interest and (ii) a binding site for an inhibitory RNA molecule; and subsequently (b) administering to the subject an inhibitory RNA molecule, wherein the inhibitory RNA molecule has complementarity to the binding site on the AAV vector, optionally wherein the recombinant AAV is administered to the subject intramuscularly.

2. METHOD FOR REDUCING EXPRESSION OR ACTIVITY OF A PROTEIN OF INTEREST IN A SUBJECT, the method characterized by comprising administering to the subject an inhibitory RNA molecule, wherein the subject has been previously administered a recombinant AAV vector comprising (i) a transgene encoding the protein of interest and (ii) a binding site having complementarity to the inhibitory RNA molecule, optionally wherein the recombinant AAV is administered to the subject intramuscularly.

3. METHOD FOR TEMPORARILY MODULATING THE EXPRESSION OR ACTIVITY OF A PROTEIN OF INTEREST IN A SUBJECT, the method characterized by comprising: (a) administering to the subject a recombinant AAV vector comprising (i) a transgene encoding the protein of interest and (ii) a binding site for an inhibitory RNA molecule, wherein administration of the recombinant AAV increases the expression or activity of the protein of interest in the subject; and subsequently Petition 870250098454, 10 / 28 / 2025, p. 150 / 170 2 / 8 (b) administering to the subject an inhibitory RNA molecule, wherein the inhibitory RNA molecule has complementarity with the binding site on the AAV vector, wherein administration of the inhibitory RNA molecule reduces the expression or activity of the protein of interest in the subject; optionally, wherein the recombinant AAV is administered to the subject intramuscularly.

4. METHOD FOR TREATING A DISEASE in a subject in need thereof, wherein the disease is one that (i) is associated with or caused by a reduction in expression or activity of a protein of interest relative to a subject without the disease, or (ii) is improved by increasing expression or activity of a protein of interest in the subject, the method characterized by comprising: (a) administering to the subject a recombinant AAV vector comprising (i) a transgene encoding a protein of interest and (ii) a binding site for an inhibitory RNA molecule; and subsequently (b) administering to the subject an inhibitory RNA molecule, wherein the inhibitory RNA molecule has complementarity to the binding site on the AAV vector, optionally wherein the recombinant AAV is administered to the subject intramuscularly.

5. METHOD FOR TREATING A DISEASE in a subject in need thereof, wherein the disease is one that (i) is associated with or caused by a reduction in expression or activity of a protein of interest relative to a subject who does not have the disease, or (ii) is improved by increasing expression or activity of a protein of interest in the subject, the method characterized by comprising administering to the subject an inhibitory RNA molecule, wherein the subject has been previously administered a recombinant AAV vector comprising (i) a transgene encoding the protein of interest and (ii) a binding site having complementarity to the inhibitory RNA molecule, optionally wherein the recombinant AAV is administered to the subject Petition 870250098454, of 10 / 28 / 2025, p. 151 / 170 3 / 8 by intramuscular route.

6. METHOD, according to any one of claims 1 to 5, characterized in that the recombinant AAV is administered to the subject between one and ten times, optionally in that the recombinant AAV is administered to the subject one, two, three, four, five, six, seven, eight, nine or ten times.

7. METHOD, according to claim 6, characterized in that the recombinant AAV is administered to the subject between one and ten times, optionally in that the recombinant AAV is administered to the subject one, two, three, four or five times.

8. METHOD, according to claim 7, characterized in that the recombinant AAV is administered to the subject between one and three times, optionally in that the recombinant AAV is administered to the subject one, two or three times.

9. METHOD, according to claim 8, characterized in that the recombinant AAV is administered to the subject once or twice.

10. METHOD, according to claim 9, characterized by the recombinant AAV being administered to the subject once.

11. METHOD, of any of claims 1 to 10, characterized by the subject having or being at risk of developing a disease listed in Table 1.

12. METHOD, according to any one of claims 1 to 11, characterized in that the transgene is a gene listed in Table 1.

13. METHOD, according to any one of claims 1 to 12, characterized in that the expression of the protein of interest is increased following administration of the recombinant AAV.

14. METHOD, according to claim 13, characterized in that the expression of the protein of interest is sustained for at least five days following administration of the recombinant AAV. Petition 870250098454, dated 10 / 28 / 2025, pp. 152 / 170 4 / 8 15. METHOD, according to claim 14, characterized in that the expression of the protein of interest is sustained for at least thirty days following administration of the recombinant AAV.

16. METHOD, according to claim 15, characterized in that the expression of the protein of interest is sustained for at least sixty days following administration of the recombinant AAV.

17. METHOD, according to claim 16, characterized in that the expression of the protein of interest is sustained for at least ninety days following administration of the recombinant AAV.

18. METHOD, according to claim 17, characterized in that the expression of the protein of interest is sustained for at least 120 days following administration of the recombinant AAV.

19. METHOD, according to claim 18, characterized in that the expression of the protein of interest is sustained for at least one year following administration of the recombinant AAV.

20. METHOD, according to claim 19, characterized in that the expression of the protein of interest is sustained for between one and five years following administration of the recombinant AAV.

21. METHOD, according to claim 20, characterized in that the expression of the protein of interest is sustained for at least 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more than 10 years following administration of the recombinant AAV.

22. METHOD, according to any one of claims 1 to 21, characterized in that the protein of interest is expressed at a therapeutic or subtherapeutic level following administration of the recombinant AAV.

23. METHOD, according to any one of claims 1 to 22, characterized in that the inhibitory RNA molecule is selected from a small interfering RNA (siRNA), an antisense oligonucleotide (ASO), a double-stranded RNA (dsRNA), a hairpin RNA (shRNA), a microRNA (miRNA) or a gapmer.

24. METHOD, according to claim 23, characterized in that the inhibitory RNA molecule is an siRNA.

25. METHOD, according to claim 23 or 24, characterized in that the inhibitory RNA molecule further comprises a selected modification of a chemically modified sugar, a chemically modified nucleobase and a chemically modified internucleoside bond.

26. METHOD, according to any one of claims 23 to 25, characterized in that the inhibitory RNA molecule further comprises a hydrophobic fraction.

27. METHOD, according to claim 26, characterized in that the hydrophobic fraction is cholesterol.

28. Method, according to any one of claims 23 to 27, characterized in that the inhibitory RNA molecule is formulated in a delivery vehicle.

29. METHOD, according to claim 28, characterized in that the delivery vehicle is a lipid-based carrier, a liposome or a lipid nanoparticle.

30. METHOD, according to any one of claims 1 to 29, characterized in that the inhibitory RNA molecule lacks sufficient complementarity to hybridize with an endogenous RNA sequence that occurs naturally in a subject cell.

31. METHOD, according to claim 30, characterized in that the inhibitory RNA molecule has complementarity with an mRNA transcript, or a portion thereof, that is transcribed from the recombinant AAV vector and that encodes the protein of interest.

32. METHOD, according to claim 31, characterized in that the inhibitory RNA molecule has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% complementarity with a region within the mRNA transcript.

33. METHOD, according to claim 32, characterized in that the inhibitory RNA molecule has 100% complementarity with the region within the mRNA transcript.

34. METHOD, according to any one of claims 31 to 33, characterized in that the inhibitory RNA molecule contains a sequence that has complementarity with at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29 or at least 30 contiguous nucleotides within the mRNA transcript.

35. METHOD, according to any one of claims 1 to 34, characterized in that the binding site of the inhibitory RNA molecule is present within the 3' untranslated region (UTR) of the transgene, the 5' UTR of the transgene, a region upstream of the start codon in the transgene, a region downstream of the stop codon in the transgene, or a region in the open reading frame of the transgene.

36. METHOD, according to any one of claims 1 to 35, characterized in that the inhibitory RNA molecule is administered intramuscularly to the subject.

37. METHOD, according to any one of claims 1 to 36, characterized by the administration of the inhibitory RNA molecule reducing the 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%, compared with the expression of the protein of interest in the subject before administration of the inhibitory RNA molecule.

38. METHOD, according to any one of claims 1 to 36, characterized by the administration of the inhibitory RNA molecule reducing the expression of the protein of interest by about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times or more than 50 times, compared with the expression of the protein of interest in the subject before administration of the inhibitory RNA molecule.

39. METHOD, according to claim 37 or 38, characterized by the administration of the inhibitory RNA molecule reducing the expression of the protein of interest for at least five days.

40. METHOD, according to claim 39, characterized by the administration of the inhibitory RNA molecule reducing the expression of the protein of interest for at least thirty days.

41. METHOD, according to claim 40, characterized by the inhibitory RNA molecule reducing the expression of the protein of interest for at least sixty days.

42. METHOD, according to claim 41, characterized by the administration of the inhibitory RNA molecule reducing the expression of the protein of interest for at least ninety days.

43. METHOD, according to claim 42, characterized by the administration of the inhibitory RNA molecule reducing the expression of the protein Petition 870250098454, dated 10 / 28 / 2025, pp. 156 / 170 8 / 8 of interest for at least 120 days.

44. METHOD, according to claim 43, characterized by the administration of the inhibitory RNA molecule reducing the expression of the protein of interest for at least one year.

45. METHOD, according to claim 44, characterized by the administration of the inhibitory RNA molecule reducing the expression of the protein of interest for at least five years.

46. ​​METHOD, according to any one of claims 1 to 45, characterized in that the AAV vector is an AAV vector of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, rh10 or rh74, or in that the AAV vector is encapsulated by one or more synthetic capsid proteins.

47. METHOD, according to any one of claims 1 to 46, characterized in that the inhibitory RNA molecule is administered to the subject before the subject receives a therapeutic intervention that is contraindicated with the protein of interest.

48. METHOD, according to claim 47, characterized in that the inhibitory RNA molecule is administered to the subject from one day to 12 months before the subject receives a therapeutic intervention that is contraindicated with the protein of interest.

49. METHOD, according to claim 48, characterized by the inhibitory RNA molecule being 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 before receiving a therapeutic intervention that is contraindicated with the protein of interest.

50. METHOD, according to any one of claims 1 to 49, characterized in that the subject is a human. Petition 870250098454, dated 10 / 28 / 2025, pp. 157 / 170

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