Products and methods for measuring the potency of a silencing gene therapy

Abstract

A host cell comprising a first vector comprising a DNA comprising a nucleotide sequence encoding an adeno-associated virus (AAV) receptor (AAVR) or a variant thereof comprising AAVR biological activity; and a second vector comprising a DNA comprising a nucleotide sequence encoding a target gene tagged with a reporter gene is provided. A cell line comprising a plurality of the host cells comprising a first vector comprising a DNA comprising a nucleotide sequence encoding an adeno-associated virus (AAV) receptor (AAVR) or a variant thereof comprising AAVR biological activity; and a second vector comprising a DNA comprising a nucleotide sequence encoding a target gene tagged with a reporter gene is provided. A method for measuring the potency of a silencing gene therapy, the method comprising: transducing a host cell comprising a first vector comprising a DNA comprising a nucleotide sequence encoding an adeno-associated virus (AAV) receptor (AAVR) or a variant thereof comprising AAVR biological activity; and a second vector comprising a DNA comprising a nucleotide sequence encoding a target gene tagged with a reporter gene with an AAV comprising a DNA comprising a nucleotide sequence that inhibits expression of the target gene; measuring reporter gene expression in the host cell and a control; and determining a level of RNA inhibition based upon the difference in reporter gene expression in the host cell and the control is provided. In some aspects, the method is used as a potency assay to measure the potency of the AAV gene therapy product and / or measure the strength and stability of a therapeutic AAV product over time and under different storage conditions.

Description

28335 / 708172024-081 -02PRODUCTS AND METHODS FOR MEASURING THE POTENCY OF A SILENCING GENE THERAPYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to US Provisional Application No. 63 / 716,014, filed on November 4, 2024, the entire contents of each of which are fully incorporated herein by reference.FIELD

[0002] Products and methods for a universal or platform approach or technology for an in vitro potency assay to measure potency of a knockdown gene therapy when AAV is used to deliver a DNA expression cassette for a genetic silencing therapy. Such platform approach or technology has the potential to be used in testing the potency for more than one gene therapy without any adverse effect on quality, manufacturing, or safety and such use of the platform approach or technology offers significant efficiencies to the development or manufacturing process for various gene therapies.BACKGROUND

[0003] Adeno-associated viruses (AAV) are a cornerstone system for delivering gene therapies for multiple types of diseases. Despite the immense progress made in the field to develop AAV-based gene therapies, many technological and manufacturing challenges remain that serve as bottlenecks to translation and contribute to the high cost for a single dose of an AAV therapy. While most AAV therapies involve gene replacement for recessive disorders, there are many dominant diseases that would benefit from disease gene silencing, including, but not limited to, knockdown gene therapy. Thus, research is ongoing for using AAV as a vector for delivering DNA cassettes expressing various types of inhibitory RNAs (RNAi) engineered to silence these dominant genes.

[0004] One aspect of this research involves the use of a potency assay to measure the potency of the AAV gene therapy product and / or measure the strength and stability of a therapeutic AAV product over time and under different storage conditions. A potency assay is a quantitative test that measures the biological activity of a product to ensure its quality, and for determining more quickly if such type of AAV-based gene therapy meets the product release specifications and is, therefore, a valuable tool during product release testing. Potency assays are used to ensure consistency of the product, identify sub-potent batches, and discriminate between product batches, but they often can take quite a bit of time and money, including time and money involved in their development. Potency assays are important during product development and manufacturing and are required for the release of new biological products, including AAV gene therapies. AAV gene therapies are highly28335 / 708172024-081 -02 specific in their mode of action and target cell types. As such, developing a potency assay for an AAV gene therapy is extremely challenging.

[0005] Thus, there is a need in the art for improved products and methods for measuring the potency of an AAV gene therapy.SUMMARY

[0006] The disclosure provides a universal or platform approach for an in vitro potency assay to measure potency of a knockdown gene therapy. An effective potency assay can measure the strength and stability of a therapeutic product over time and under different storage conditions, making it a valuable tool during product release testing. Thus, the disclosure provides a universal or platform approach for carrying out a potency assay to assess the potency of knockdown when AAV is used to deliver a DNA expression cassette for a genetic silencing therapy. Such platform approach or platform technology has the potential to be used in testing the potency for more than one gene therapy without any adverse effect on quality, manufacturing, or safety and that the use of the platform technology would bring significant efficiencies to the development or manufacturing process for the gene therapy.

[0007] An effective potency assay can measure the strength and stability of a therapeutic product over time and under different storage conditions, making it a valuable tool during product release testing. The disclosure provides products and methods useful in a universal method for assessing potency of AAVs delivering inhibitory RNAs, including but not limited to miRNAs. Thus, the disclosure provides host cells, stable cell lines, and methods for measuring the potency of a silencing gene therapy.

[0008] AAVs are a cornerstone system for delivering gene therapies for several diseases, including two under development, as discussed herein, Charcot-Marie-Tooth disease type 1A (CMT1A) resulting from aberrant expression of the PMP22 gene, and Facioscapulohumeral muscular dystrophy (FSHD) resulting from aberrant expression of the DUX4 gene. While most AAV therapies today involve gene replacement for recessive disorders, CMT1A and FSHD are dominant diseases that would benefit from disease gene silencing, for which extensive pre-clinical safety and efficacy data to support translating gene therapies for both diseases have been developed. Thus, the disclosure provides products and methods for carrying out a robust potency assay to assess product strength and stability. Thus, the disclosure provides a detailed description for creating stable cell lines containing (1) the AAV receptor (AAVR) to improve AAV transduction, and (2) a Renilla luciferase (rLuc) open reading frame with disease gene sequences in the 3’ UTR, to enable disease gene knockdown quantification. The disclosure thus provides a straightforward28335 / 708172024-081 -02 framework for potency assay development supporting AAV-mediated and non-viral gene silencing programs. The disclosure thus provides a versatile and easy-to-implement in vitro, cell-based potency assay for any gene silencing therapy. Although the disclosure exemplifies products and methods for measuring potency of any viral or non-viral gene silencing product targeting PMP22 or DLIX4 mRNA, i.e., methodology for in vitro potency assay development to support CMT1A and FSHD gene therapies, the products and methods described herein are intended to be applicable for measuring potency of any viral or non- viral gene silencing product targeting other mRNAs for other gene therapies as well.

[0009] Provided herein, therefore, is a host cell comprising a first vector comprising a DNA comprising a nucleotide sequence encoding an adeno-associated virus (AAV) receptor (AAVR) or a variant thereof comprising AAVR biological activity; and a second vector comprising a DNA comprising a nucleotide sequence encoding a target gene tagged with a reporter gene. In some aspects, the first vector and / or the second vector is a lentiviral vector. In some aspects, the first vector is a lentiviral vector and the second vector is a lentiviral vector. In some aspects, the target gene is a gene desired to be silenced or knocked down. In some aspects, the gene desired to be silenced or knocked down is a gene in which its expression or overexpression is associated with a disease or disorder. In some aspects, the first vector and / or the second vector further comprises an antibiotic resistant gene. In some aspects, the antibiotic resistance gene of the first vector and the antibiotic resistance gene of the second vector are different. In some aspects, the antibiotic resistance gene is a puromycin resistance gene, a neomycin resistance gene, a blasticidin resistance gene, a hygromycin resistance gene, a mycophenolic acid resistance gene, or a zeocin resistance gene. In some aspects, the antibiotic resistance gene of first vector is a puromycin resistance gene. In some aspects, the antibiotic resistance gene of the second vector is a neomycin resistance gene or a blasticidin resistance gene. In some aspects, the first vector and / or the second vector further comprises a promoter. In various aspects, the promoter or enhancer may be a ubiquitous promoter or enhancer. In some aspects, a promoter that targets the gene of interest to certain cell types is used. In some aspects, the promoter is any of LI6, LI7, tRNA, H1 , CMV, minimal CMV, T7, EF1 -alpha, Minimal EF1 -alpha, or a tissue-specific promoter including, but not limited to, a muscle-specific promoter, a neurospecific promoter, a Schwann cell-specific or myelin-specific promoter, or a cardiacspecific promoter. In some aspects, the promoter is CMV, U6 or H1 . In some aspects, the muscle-specific promoter is unc45b, muscle creatine kinase (MCK), tMCK, minimal MCK, CK6, CK7, CK8, alpha-myosin heavy chain enhancer- / MCK enhancer-promoter (MHCK7), or CK1. In some aspects, the myelin-specific promoter is myelin specific protein-Z (MPZ, P0), peripheral myelin protein, 22 (PMP22), myelin basic protein (MBP), or SRY (sex determining28335 / 708172024-081 -02 region Y)-box 10 protein (SOX10). In some aspects, the cardiac-specific promoter is alphamyosin heavy chain enhancer- / MCK enhancer-promoter (MHCK7), the 250-bp fragment of the myosin light chain-2v (MLC-2v) gene promoter (MLC250), cardiac troponin T (cTnT) promoter, the a-myosin heavy chain (a-MHC) promoter, muscle creatine kinase (MCK), tMCK, minimal MCK, CK6, CK7, CK8, or CK1 . In some aspects, the reporter gene is a gene encoding a luciferase, a fluorescent protein, a LacZ, or a beta-lactamase. In some aspects, the reporter gene is a gene encoding a luciferase. In some aspects, the luciferase is a Renilla luciferase. In some aspects, the cell is a human embryonic kidney (HEK) cell, a human alveolar basal epithelial cell (A549), a HeLa cell, a Chinese hamster ovary (CHO) cell, or a myoblast cell (C2C12). In some aspects, the HEK cell is a HEK293 cell. In some aspects, the DNA comprising the nucleotide sequence encoding the target gene tagged with the reporter gene is positioned or designed in the vector gene so that the target gene is not transcribed or translated in the cell, whereas the reporter gene tagged to the target gene is transcribed and / or translated in the cell.

[0010] The disclosure provides a cell line comprising a plurality of the host cells comprising a first vector comprising a DNA comprising a nucleotide sequence encoding an adeno-associated virus (AAV) receptor (AAVR) or a variant thereof comprising AAVR biological activity; and a second vector comprising a DNA comprising a nucleotide sequence encoding a target gene tagged with a reporter gene. In some aspects, the cell line is selected for its ability to grow in media comprising at least one antibiotic, wherein the at least one antibiotic is the antibiotic of the antibiotic resistance gene present in the first and / or second vector. In some aspects, the host cell and / or the cell line is grown in the presence of at least two or more antibiotics, wherein the antibiotic present in the media is selected as having relevance to the antibiotic-resistance gene of the vector or plasmid. In some aspects, the first vector and / or the second vector is a lentiviral vector. In some aspects, the first vector is a lentiviral vector and the second vector is a lentiviral vector. In some aspects, the target gene is a gene desired to be silenced or knocked down. In some aspects, the gene desired to be silenced or knocked down is a gene in which its expression or overexpression is associated with a disease or disorder. In some aspects, the first vector and / or the second vector further comprises an antibiotic resistant gene. In some aspects, the antibiotic resistance gene of the first vector and the antibiotic resistance gene of the second vector are different. In some aspects, the antibiotic resistance gene is a puromycin resistance gene, a neomycin resistance gene, a blasticidin resistance gene, a hygromycin resistance gene, a mycophenolic acid resistance gene, or a zeocin resistance gene. In some aspects, the antibiotic resistance gene of first vector is a puromycin resistance gene. In some aspects, the antibiotic resistance gene of the second vector is a neomycin resistance gene or a28335 / 708172024-081 -02 blasticidin resistance gene. In some aspects, the first vector and / or the second vector further comprises a promoter. In various aspects, the promoter or enhancer may be a ubiquitous promoter or enhancer. In some aspects, a promoter that targets the gene of interest to certain cell types is used. In some aspects, the promoter is any of U6, U7, tRNA, H1 , CMV, minimal CMV, T7, EF1 -alpha, Minimal EF1 -alpha, or a tissue-specific promoter including, but not limited to, a muscle-specific promoter, a neurospecific promoter, a Schwann cell-specific or myelin-specific promoter, or a cardiac-specific promoter. In some aspects, the promoter is CMV, U6 or H1 . In some aspects, the muscle-specific promoter is unc45b, muscle creatine kinase (MCK), tMCK, minimal MCK, CK6, CK7, CK8, alpha-myosin heavy chain enhancer- / MCK enhancer-promoter (MHCK7), or CK1 . In some aspects, the myelin-specific promoter is myelin specific protein-Z (MPZ, P0), peripheral myelin protein, 22 (PMP22), myelin basic protein (MBP), or SRY (sex determining region Y)-box 10 protein (SOX10). In some aspects, the cardiac-specific promoter is alpha-myosin heavy chain enhancer- / MCK enhancer-promoter (MHCK7), the 250-bp fragment of the myosin light chain-2v (MLC-2v) gene promoter (MLC250), cardiac troponin T (cTnT) promoter, the a-myosin heavy chain (a- MHC) promoter, muscle creatine kinase (MCK), tMCK, minimal MCK, CK6, CK7, CK8, or CK1 . In some aspects, the reporter gene is a gene encoding a luciferase, a fluorescent protein, a LacZ, or a beta-lactamase. In some aspects, the reporter gene is a gene encoding a luciferase. In some aspects, the luciferase is a Renilla luciferase. In some aspects, the cell is a human embryonic kidney (HEK) cell, a human alveolar basal epithelial cell (A549), a HeLa cell, a Chinese hamster ovary (CHO) cell, or a myoblast cell (C2C12). In some aspects, the HEK cell is a HEK293 cell. In some aspects, the DNA comprising the nucleotide sequence encoding the target gene tagged with the reporter gene is positioned or designed in the vector gene so that the target gene is not transcribed or translated in the cell, whereas the reporter gene tagged to the target gene is transcribed and / or translated in the cell.

[0011] The disclosure provides a method for measuring the potency of a silencing gene therapy, the method comprising: transducing a host cell comprising a first vector comprising a DNA comprising a nucleotide sequence encoding an adeno-associated virus (AAV) receptor (AAVR) or a variant thereof comprising AAVR biological activity; and a second vector comprising a DNA comprising a nucleotide sequence encoding a target gene tagged with a reporter gene with an AAV comprising a DNA comprising a nucleotide sequence encoding an inhibitory RNA designed to inhibit expression of the target gene; measuring reporter gene expression in the host cell and a control; and determining a level of RNA inhibition based upon the difference in reporter gene expression in the host cell and the control. In some aspects, the level of RNA inhibition is a measure of the potency of the28335 / 708172024-081 -02 silencing gene therapy. In some aspects, the first vector and / or the second vector is a lentiviral vector. In some aspects, the first vector is a lentiviral vector and the second vector is a lentiviral vector. In some aspects, the target gene is a gene desired to be silenced or knocked down. In some aspects, the gene desired to be silenced or knocked down is a gene in which its expression or overexpression is associated with a disease or disorder. In some aspects, the first vector and / or the second vector further comprises an antibiotic resistant gene. In some aspects, the antibiotic resistance gene of the first vector and the antibiotic resistance gene of the second vector are different. In some aspects, the antibiotic resistance gene is a puromycin resistance gene, a neomycin resistance gene, a blasticidin resistance gene, a hygromycin resistance gene, a mycophenolic acid resistance gene, or a zeocin resistance gene. In some aspects, the antibiotic resistance gene of first vector is a puromycin resistance gene. In some aspects, the antibiotic resistance gene of the second vector is a neomycin resistance gene or a blasticidin resistance gene. In some aspects, the first vector and / or the second vector further comprises a promoter. In various aspects, the promoter or enhancer may be a ubiquitous promoter or enhancer. In some aspects, a promoter that targets the gene of interest to certain cell types is used. In some aspects, the promoter is any of LI6, LI7, tRNA, H1 , CMV, minimal CMV, T7, EF1 -alpha, Minimal EF1- alpha, or a tissue-specific promoter including, but not limited to, a muscle-specific promoter, a neurospecific promoter, a Schwann cell-specific or myelin-specific promoter, or a cardiacspecific promoter. In some aspects, the promoter is CMV, U6 or H1 . In some aspects, the muscle-specific promoter is unc45b, muscle creatine kinase (MCK), tMCK, minimal MCK, CK6, CK7, CK8, alpha-myosin heavy chain enhancer- / MCK enhancer-promoter (MHCK7), or CK1. In some aspects, the myelin-specific promoter is myelin specific protein-Z (MPZ, P0), peripheral myelin protein, 22 (PMP22), myelin basic protein (MBP), or SRY (sex determining region Y)-box 10 protein (SOX10). In some aspects, the cardiac-specific promoter is alphamyosin heavy chain enhancer- / MCK enhancer-promoter (MHCK7), the 250-bp fragment of the myosin light chain-2v (MLC-2v) gene promoter (MLC250), cardiac troponin T (cTnT) promoter, the a-myosin heavy chain (a-MHC) promoter, muscle creatine kinase (MCK), tMCK, minimal MCK, CK6, CK7, CK8, or CK1 . In some aspects, the reporter gene is a gene encoding a luciferase, a fluorescent protein, a LacZ, or a beta-lactamase. In some aspects, the reporter gene is a gene encoding a luciferase. In some aspects, the luciferase is a Renilla luciferase. In some aspects, the cell is a human embryonic kidney (HEK) cell, a human alveolar basal epithelial cell (A549), a HeLa cell, a Chinese hamster ovary (CHO) cell, or a myoblast cell (C2C12). In some aspects, the HEK cell is a HEK293 cell. In some aspects, the DNA comprising the nucleotide sequence encoding the target gene tagged with the reporter gene is positioned or designed in the vector gene so that the target gene is not transcribed or translated in the cell, whereas the reporter gene tagged to the target gene is28335 / 708172024-081 -02 transcribed and / or translated in the cell. In some aspects, the inhibitory RNA is a microRNA (miRNA), a short inhibitory RNA (siRNA), or a short hairpin RNA (shRNA). In some aspects, the inhibitory RNA is a miRNA. In some aspects, the inhibitory RNA is a miRNA that targets DUX4 or PMP22. In some aspects, the method is used as a potency assay to measure the potency of the AAV gene therapy product and / or measure the strength and stability of a therapeutic AAV product over time and under different storage conditions. In some aspects, the AAV is AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh74, AAV.rh8, AAV.rh , AAV11 , AAV12, AAV13, AAV-anc80, AAV-B1 , AAV-BR1 , AAV.PHP.EB, AAVv66, AAV2 / 1 , AAV2 / 8, or AAV2 / 9, AAVMYO, MYOAAV, MYOAAV1A, MYOAAV2A, MYOAAV3A, modified AAV9 (mAAV9), or AAV-SLB101 , or any derivative thereof, or any modified AAV, chimeric AAV, or variant thereof.

[0012] In some aspects, the disclosure provides a method for measuring the potency of a silencing gene therapy, the method comprising transducing a host cell with an AAV comprising a nucleotide sequence that inhibits expression of the target gene; measuring reporter gene expression in the host cell and a control; and determining a level of RNA inhibition based upon the difference in reporter gene expression in the host cell and the control.

[0013] In some aspects, the host cell comprises a first vector comprising a DNA comprising a nucleotide sequence encoding an adeno-associated virus (AAV) receptor (AAVR) or a variant thereof comprising AAVR biological activity; and a second vector comprising a DNA comprising a nucleotide sequence encoding a target gene tagged with a reporter gene. In some aspects, the first vector or the second vector is a lentiviral vector. In some aspects, the first vector is a lentiviral vector and the second vector is a lentiviral vector. In some aspects, the target gene is a gene desired to be silenced or knocked down. In some aspects, the gene desired to be silenced or knocked down is a gene in which its expression or overexpression is associated with a disease or disorder. In some aspects, the target gene is PMP22or DUX4. In some aspects, the first vector and / or the second vector further comprises an antibiotic resistant gene. In some aspects, the antibiotic resistance gene of the first vector and the antibiotic resistance gene of the second vector are different. In some aspects, the antibiotic resistance gene is a puromycin resistance gene, a neomycin resistance gene, a blasticidin resistance gene, a hygromycin resistance gene, a mycophenolic acid resistance gene, or a zeocin resistance gene. In some aspects, the antibiotic resistance gene of first vector is a puromycin resistance gene. In some aspects, the antibiotic resistance gene of the second vector is a neomycin resistance gene or a blasticidin resistance gene. In some aspects, the first vector and / or the second vector further comprises a promoter. In some aspects, the reporter gene is a gene encoding a luciferase, a28335 / 708172024-081 -02 fluorescent protein, a LacZ, or a beta-lactamase. In some aspects, the reporter gene is a gene encoding a luciferase. In some aspects, the luciferase is a Renilla luciferase. In some aspects, the cell is a human embryonic kidney (HEK) cell, a human alveolar basal epithelial cell (A549), a HeLa cell, a Chinese hamster ovary (CHO) cell, or a myoblast cell (C2C12). In some aspects, the HEK cell is a HEK293 cell. In some aspects, the DNA comprising the nucleotide sequence encoding the target gene tagged with the reporter gene is positioned or designed in the vector gene so that the target gene is not transcribed or translated in the cell, whereas the reporter gene tagged to the target gene is transcribed and / or translated in the cell.

[0014] In some aspects, the level of RNA inhibition is a measure of the potency of the silencing gene therapy. In some aspects, the nucleotide sequence that inhibits expression of the target gene is a nucleotide sequence encoding a microRNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense oligonucleotide, morpholino oligonucleotide, ribozyme, or CRISPR interference (CRISPRi) components. In some aspects, the nucleotide sequence that inhibits expression of the target gene is a microRNA (miRNA), a short inhibitory RNA (siRNA), or a short hairpin RNA (shRNA). In some aspects, the nucleotide sequence that inhibits expression of the target gene is a nucleotide sequence encoding a microRNA. In some aspects, the nucleotide sequence that inhibits expression of the target gene is a nucleotide sequence that targets DUX4 or PMP22. In some aspects, the microRNA is a DLIX4 or a PMP22 microRNA and / or the target gene is DUX4 or PMP22.

[0015] In some aspects, the method is used as a potency assay to measure the potency of the AAV gene therapy product and / or measure the strength and stability of a therapeutic AAV product over time and under different storage conditions.

[0016] In some aspects, the AAV is AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh74, AAV.rh8, AAV.rhW, AAV11 , AAV12, AAV13, AAV-anc80, AAV-B1 , AAV-BR1 , AAV.PHP.EB, AAVv66, AAV2 / 1 , AAV2 / 8, or AAV2 / 9, AAVMYO, MYOAAV, MYOAAV1A, MYOAAV2A, MYOAAV3A, modified AAV9 (mAAV9), or AAV-SLB101 , or any derivative thereof, or any modified AAV, chimeric AAV, or variant thereof.

[0017] Further aspects and advantages of the disclosure will be apparent to those of ordinary skill in the art from a review of the following detailed description, taken in conjunction with the drawings. It should be understood, however, that the detailed description (including the drawings and the specific examples), while indicating embodiments of the disclosed subject matter, are given by way of illustration only, because various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.28335 / 708172024-081 -02BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Fig. 1 A-B provides a schematic overview of the stable cell line generation and design. Fig. 1 A shows a stable cell line containing two components, the AAV Receptor (AAVR), and a Renilla tagged target gene. The target gene is the gene of interest or the gene to be silenced, e.g., in the working Examples, PMP22 or DUX4. Fig. 1 B shows the treatment of a stable cell line with a therapeutic AAV. AAV utilizes AAVR for improved transduction. The AAV genome containing a miRNA expression cassette is translocated to the nucleus and following expression becomes processed and loaded into the RISC complex and used to degrade the Ren / 7 / a-Target Gene transcript resulting in a decrease in Renilla expression in the Renilla- Luciferase assay. After AAV treatment, miRNAs are transcribed in the host cell nucleus, processed by RNAi machinery, and loaded into the RNA-induced silencing complex (RISC) to trigger target transcript degradation and rLuc signal reduction.

[0019] Fig. 2 provides a flow chart schematic of a pipeline for stable cell line creation, selection, clonal isolation, expansion, and testing. Top plasmid, AAVR.flag-PuroR. Bottom plasmid, schematic represents either rLucPMP22-NeoR or rLucDUX4-BlastR. AbR, antibiotic resistance gene, where AbR is neomycin resistance (NeoR) or Blasticidin resistance (BlastR) as indicated. Cells were produced using dual lentiviral transduction on a 6-well plate followed by antibiotic selection and single cell isolation. Clones tested for AAVR. Flag expression, improved AAV transduction, and rLuc knockdown after treatment.

[0020] Fig. 3A-B shows data from some stable cell line tests screened for the presence of Renilla luciferase (rLUC) and AAVR. Flag expression. Fig. 3A shows the amount of Renilla luciferase (RLU = relative light units (RLU) measured after 50,000 cells of 22 different PMP22 clones were plated in a 96-well plate, and 24hrs later media was removed, cells were lysed, and the Renilla luciferase signal was measured. Fig. 3B shows an anti-Flag western blot of protein lysates from Mol 5 E5 stable cells and HEK293T cells normalized by cell count during Radio-Immunoprecipitation Assay (RIPA) lyses and loaded onto SDS- PAGE under reducing conditions, and probed with an HRP-conjugated anti-flag antibody. Clone Mol 5 E5 expressed the flag-tagged AAVR protein. AAVR has a predicted molecular weight of 108 kDa, but due to heavy glycosylation, migrates at -150 kDa, as seen here.

[0021] Fig. 4A-C shows improved scAAV9-EFGP transduction and quantifiable Renilla luciferase-PMP22 knockdown by scAAV9-miR871 . Fig. 4A shows fluorescent microscopy of HEK293T and stable cell line 24hrs post-transduction by scAAV9-EGFP. Fig. 4B shows quantified GFP expression 48hrs after scAAV9-EGFP transduction; significance determined using multiple t-test; p-value<0.005. Fig. 4C shows Renilla luciferase knockdown following28335 / 708172024-081 -02 scAAV9-miR871 transduction. Data are displayed as mean relative light units (RLU) ±SD with at least 3 technical and experimental replicates.

[0022] Fig. 5A-B shows data for experiments carried out with a preliminary FSHD stable cell line. Fig. 5A show relative light units (RLU) measured after 20,000 stable cell line clones containing AAVR and Renilla luciferase-DUX4 fusion were seeded in a 96-well plate; stable cells were incubated for 2hrs; and the Renilla luciferase signal was measured in the various cell lines. Fig. 5B shows percent RLU measured by a Renilla luciferase assay after 10,000 cells were transduced by either scAAV9-mi405 or scSLBI 01 -mi405 for 24hrs at indicated Mols.

[0023] Fig. 6A-D shows results of screening for rLuc and AAVR. flag expression. Fig. 6A shows relative light units (RLU) produced from 40,000 cells of 22 different PMP22 (or CMT1 A) clones plated in a 96-well format. Only one NeoR / PuroR-resistant CMT1 A line (clone C4CMT1A) produced Renilla luciferase expression. Fig. 6B shows that clone C4CMT1Aexpresses the flag-tagged AAVR protein. Image shows a western blot of protein lysates from clone C4CMT1Aor control HEK293T cells, probed with an HRP-conjugated anti-flag antibody. AAVR has a predicted molecular weight of 108 kDa, but due to heavy glycosylation, migrates at -150 kDa, as seen here. Fig. 6C shows relative light units (RLU) produced from 40,000 cells of 21 different FSHD clones plated in a 96-well format. 1 1 of 21 clones showed luciferase expression above background. Fig. 6D shows that clones C4FSHDand C6FSHDover-express the AAVR protein, as shown by western blot, similar to 6B. Bar Graphs display mean (SD) using experimental and biological triplicates.

[0024] Fig. 7A-B shows results of an assessment of AAV vector transduction in C4CMT1A, C4FSHD, and C6FSHDcell lines. 10,000 cells were plated in a 96-well format and transduced by AAV9, SLB101 , MYOAAV3A, MYOAAV2A, or AAV6 expressing CMV.GFP genomes. Fig. 7A shows fluorescent microscopy images which demonstrate visual extent of the transduction 24hrs after transduction. Fig. 7B shows quantification of GFP signal 48hrs after transduction using a GloMax plate reader and plotted as % RFU (relative fluorescent units) compared to untransduced HEK293T cells. Data represent mean (SD), with significance determined using non-parametric t-test vs 293T cells, **** p <0.0001 , *** p<0.005, ** p<0.01 , * p<0.05, ns p>0.05.

[0025] Fig. 8A-D shows that stable cell lines are useful for CMT1A and FSHD gene therapy potency assays. Fig. 8A shows dose-dependent, treatment-specific knockdown of rLuc activity in C4CMT1A, C4FSHD, and C6FSHDstable cells treated with two doses of therapeutic AAV. For CMT1A, scAAV9.mi871 ; for FSHD, SLB101 ,mi405. Fig. 8B-C shows that potency assays identify defective or non-functional AAV vectors. C4CMT1Aand C4FSHDtreated with28335 / 708172024-081 -02 indicated vectors with and without multiple cycles of freeze-thaw (1x, 3x, 5x, 10x) or boiling (98QC for 5 minutes). SLB101 ,mi405 served as negative control for C4CMT1Acells and AAV9.miR871 served as negative control for C4FSHDcells. Lack of rLuc knockdown by negative controls in both assays supports assay specificity. Fig. 8D shows that C4FSHDcells detect DUX4-targeted silencing beyond mi405. C4FSHDcells were transduced by AAV6 vectors expressing mi405, a control mi405 lacking a terminator sequence (mi405-No T6), or two additional vectors expressing artificial miRNA that bind and silence DUX4 at different locations (mi333, mil 85). RLU values represent mean (SD), with significance determined using one-way ANOVA, **** p <0.0001 , *** p<0.005, ** p<0.01 , ns p>0.05.

[0026] Fig. 9A-D shows results of screening FSHD stable cell lines. Fig. 9A-B shows results of testing dose-dependent knockdown of rLuc activity in FSHD cell lines 24hrs following treatment with scAAV9.mi405 (A) or scSLB101 ,mi405 (B) with an MOI of 5e6, 1 e6 and 2e5. FSHD stable cell lines narrowed down to 5 candidates following A-B rLuc assay. Fig. 8C shows rLuc knockdown 24hrs after scSLB101 ,mi405 treatment with an MOI of 8e6 and 1 .6e6. Fig. 9D shows rLuc knockdown 48hrs after scSLBI 01 ,mi405 treatment with an MOI of 8e6 and 1 .6e6. C4 and C6 were selected for additional testing due to significant dose response at 48hrs after scSLBI 01 ,mi405 treatment. Preliminary screen was conducted only once to narrow down candidate with experimental triplicate and plotted as mean (SD) using Graph Pad 10.DETAILED DESCRIPTION

[0027] Nearly 30 viral and non-viral in vivo gene therapy products are now FDA-approved for use in the United States, with various analysts predicting many more products to be approved by 2030. Rapidly evolving gene therapy technology, such as engineered AAV capsids or development of safer, in vivo gene editing approaches, could also expand the field by providing tools to treat disease targets for which gene therapy was not previously indicated. Prior to clinical use, each program requires development of a potency assay, which typically needs to be uniquely designed on a drug-by-drug basis, potentially taking valuable time and resources.

[0028] The disclosure provides a novel approach to improve the efficiency of potency assay development for release testing on AAVs harboring gene knockdown machinery. The disclosure solves the problem of improving the efficiency of potency assay development for gene silencing therapies using dual lentiviral transduction to generate AAV-permissible stable cell lines and a modular, gene-specific, quantifiable luciferase readout of product potency (i.e. gene silencing). This platform approach will expedite potency assay development and regulatory approval for viral and non-viral gene silencing therapies. To28335 / 708172024-081 -02 ensure simplicity and avoid complications of using animal models, a cell based in vitro potency assay was developed.

[0029] A potency assay is used to ensure lot-to-lot consistency in AAV manufacturing, and thus also plays a valuable role in comparability studies and stability testing (Addressing potency-assay related development delays for cell and gene therapies: Results of a scientific exchange between FDA and developers; March 30, 2023; asgct.org / advocacy / policy- statements / addressing-potency-assay-related-development-delays-for-cell-and-gene- therapies-results-of-a-scientific-exchange-between-fda-and-developers). During therapeutic development, prior to clinical application, a potency assay is a valuable tool to assess AAV manufacturing consistency, or variances that could impact the strength of the final product. As such, regulatory agencies recommend designing an appropriate potency assay as soon as possible, to ensure product potency across the entire drug development pipeline (13). Potency is defined by the FDA as “the specific ability or capacity of the product to effect a given result” (21 CFR 600.3(s)) (Administration USFaD. Title 21 , Chapter 1 , Subchapter F - Biologies, Part 600, Subpart A, 600.3 Definitions. In: Services DoHaH, editor.: United States Food and Drug Administration; July 10, 2025).

[0030] Potency measurements may be relatively straightforward for some therapies, for example, using cell-free methods to determine binding affinity of therapeutic antibodies to a desired target (Register et al., Int J Mol Sci. 2021 ;22(10)). In contrast, gene therapy potency assay development is arguably more complex because it requires a biological system and involves a multi-step process to produce a quantifiable therapeutic effect inside a target cell. Specifically, AAV vectors must first bind a host cell, translocate across the cell membrane, escape the trans Golgi network, transport to the nucleus, and uncoat the therapeutic DNA genome where host transcriptional machinery can produce the desired therapeutic gene product (Daya et al., Clin Microbiol Rev. 2008;21 (4):583-93). Thus, gene therapies cannot be adequately tested in a cell-free system. While FDA guidance states that in vivo potency assays using animal models are acceptable for gene therapy programs, in vitro assays that limit the use of animals are encouraged. The disclosure thus provides products and methodology for an easy-to-implement in vitro, cell-based potency assay for various gene silencing therapies. The cells and assays described herein should be applicable for measuring potency of any viral or non-viral gene silencing product targeting an mRNA to silence expression of a gene.

[0031] To exemplify the products required and the methods of measuring potency as described herein, stable cell lines comprising two critical components: 1) the essential AAV receptor (AAVR) to improve AAV transduction efficiency; and 2) the target gene or disease gene of interest, e.g., PMP22 or DUX4, the gene desired to be silenced (or knocked down)28335 / 708172024-081 -02 or the gene being targeted by the inhibitory RNA (e.g., miRNA or RNAi), tagged with a reporter gene were made. Gene silencing is the process of suppressing the expression of a specific gene, essentially turning it "off" to stop it from producing a protein. Silencing is accomplished using any methods known to downregulate or knock down gene expression, Such methods include, but are not limited to, microRNAs, RNA interference (RNAi) using small interfering RNA (siRNA) or short hairpin RNA (shRNA), antisense oligonucleotides, morpholino oligonucleotides (“morpholinos”), ribozymes, and CRISPR-based approaches like CRISPR interference (CRISPRi).

[0032] In various aspects, the stable cell line is a cell line comprising a host cell comprising a first vector comprising a DNA comprising a nucleotide sequence encoding an adeno-associated virus (AAV) receptor (AAVR) or a variant thereof comprising AAVR biological activity; and a second vector comprising a DNA comprising a nucleotide sequence encoding a target gene tagged with a reporter gene.

[0033] In some aspects, the disclosure provides a method for measuring the potency of a silencing gene therapy, the method comprising transducing the host cell as described herein with an AAV comprising a nucleotide sequence that inhibits expression of the target gene; measuring reporter gene expression in the host cell and a control; and determining a level of inhibition of gene expression based upon the difference in reporter gene expression in the host cell and the control. In some aspects the inhibition of gene expression is the inhibition of RNA expression. In some aspects, the level of inhibition of gene expression is the level of RNA inhibition. In some aspects, the nucleotide sequence that inhibits expression of the target gene is “silencing” the gene, or “knocking down” expression of the gene.

[0034] In exemplary aspects, the disclosure provides products and methods for silencing, i.e., knocking down the expression of, the PMP22 and DUX4 genes. PMP22 is a protein that is a major component of the myelin sheath, the protective layer around peripheral nerves, produced by Schwann cells. Mutations in the PMP22 gene can lead to peripheral neuropathies like Charcot-Marie-Tooth disease type 1A (CMT1 A), which is caused by gene duplication, and Hereditary Neuropathy with liability to pressure palsies (HNPP), which is caused by a deletion. DUX4 is a gene located near the end of chromosome 4 that produces a protein involved in development, and its misexpression in skeletal muscle is the cause of facioscapulohumeral muscular dystrophy (FSHD). This protein is normally silenced in most adult tissues but is reactivated in individuals with FSHD, leading to muscle weakness and degeneration. DUX4 can also be involved in certain cancers.

[0035] The PMP22 / CMT 1 A and DUX4 / FSHD gene therapy products described herein effect a given result by silencing PMP22 or DLIX4 mRNAs, respectively, following delivery by28335 / 708172024-081 -02AAV vectors (Stavrou et al., J Clin Invest. 2022;132(13); Wallace et al., Mol Ther. 2012; 20(7):1417-23; Wallace et al., Mol Ther Methods Clin Dev. 2018; 8:121-30). Mechanistically, vectors for both silencing PMP22 and DLIX4 carry engineered miRNA expression cassettes (miR871 for PMP22 / CMT1 A (Stavrou et al., J Clin Invest. 2022 Jul 1 ;132(13):e159814. doi: 10.1172 / JC1159814; mi405 for DUX4 / FSHD; Wallace et al., Mol Ther. 2012;20(7):1417-23), driven by the LI6 promoter, designed to trigger RNA interference (RNAi) against the PMP22 or DUX4 mRNA. Silencing of PMP22 or DLIX4 mRNA leads to reduction of toxic PMP22 or DUX4 protein levels in Schwann cells (CMT1A) or skeletal muscle (FSHD) (Stavrou et al., J Clin Invest. 2022;132(13); Wallace et al., Mol Ther. 2012;20(7):1417-23; Wallace et al., Mol Ther Methods Clin Dev. 2018;8:121-30).

[0036] Potency measurements may be relatively straightforward for some therapies, for example, using cell-free methods to determine binding affinity of therapeutic antibodies to a desired target (15). In contrast, gene therapy potency assay development is arguably more complex because it requires a biological system and involves a multi-step process to produce a quantifiable therapeutic effect inside a target cell. Specifically, AAV vectors must first bind a host cell, translocate across the cell membrane, escape the trans Golgi network, transport to the nucleus, and uncoat the therapeutic DNA genome where host transcriptional machinery can produce the desired therapeutic gene product (16). Thus, gene therapies cannot be adequately tested in a cell-free system. While FDA guidance states that in vivo potency assays using animal models are acceptable for gene therapy programs, in vitro assays that limit the use of animals are encouraged (13, 17). In this study, we aimed to create versatile and easy-to-implement in vitro, cell-based potency assays for gene silencing therapies. The cells and assays described should be applicable for measuring potency of any viral or non-viral gene silencing product targeting PMP22 or DLIX4 mRNA.

[0037] In one aspect, a stable cell line, e.g., a HEK293T cell line, is created using dual lentiviral transduction to integrate two genetic components into the cells of the cell line: 1) the essential AAV receptor (AAVR) (the KIAA0319L gene (KIAA0319L Gene; HGNC: 30071 NCBI Gene: 79932; Ensembl: ENSG00000142687; OMIM®: 613535; UniProtKB / Swiss-Prot: Q8IZA0)) encodes a protein also known as the universal AAV receptor (AAVR)), and 2) a gene of interest tagged to a reporter gene, e.g. a Renilla Luciferase reporter gene. These two genetic components allow for improved AAV transduction and provide a quantifiable readout of gene knockdown. This cell line can then be used in a platform approach to test potency of a wide array of AAV serotypes and gene targets for therapeutic AAV release testing.

[0038] One of the advantages of the products and methods of the disclosure is that this new technology alleviates the time required for potency assay development. AAVs have a28335 / 708172024-081 -02 wide tropism and selecting a cell line can be challenging and time consuming. Incorporating the AAV receptor (AAVR) into a cell line, such as, for example, the easy-to-use HEK293T cell line, allows the resulting cell line to be more easily transduced by AAV, removing a critical first step when developing a suitable potency assay. In addition, the use of the Renilla tagged gene of interest provides a straightforward method for quantifying gene knockdown. Typically, to measure gene knockdown PCR, Western Blot, or ELISA assays would need to be used to test potency, and these tests require far more time-consuming optimization. Overall, the disclosed products and methods save time and money in executing a potency assay.

[0039] Potency assays are an essential component of FDA filing for new therapies. A potency assay is a quantitative test that measures the biological activity of a product to ensure its quality. Potency tests, along with other tests, are performed as part of product conformance testing, comparability studies, and stability testing, and they are used to ensure that a proper amount of product is delivered. Consequently, potency assays are important during product development and manufacturing and are required for the release of new biological products, including AAV gene therapies. AAV gene therapies are highly specific in their mode of action and target cell types. As such, developing a potency assay for an AAV gene therapy is extremely challenging. The disclosure provides a new approach, i.e., new products and methods, allowing a simple and efficient method to test the potency of a silencing gene therapy.

[0040] AAV or AAV vectors, as they are used interchangeably herein, are commonly used to deliver gene therapies for a wide range of diseases. Despite the immense progress made in the field to develop AAV based gene therapies, many technological and manufacturing challenges remain that serve as bottlenecks to translation and contribute to the high price tag for a single dose of an AAV therapy. While most AAV therapies involve gene replacement for recessive disorders, several dominant diseases would benefit from disease gene silencing. Specifically, many scientists are developing AAVs to deliver DNA cassettes expressing gene silencing therapies including, but not limited to, artificial microRNAs (miRNAs) engineered to silence aberrantly expressed or mutated genes. For a therapy to reach a patient population several regulatory hurdles must be cleared for FDA approval. One such hurdle is the need for a reliable potency assay to test vector strength and stability over time and between clinical batches for release testing. Developing a potency assay can be an arduous process requiring extensive optimization. The disclosure provides a universal pipeline approach / method for developing an in vitro potency assay for testing potency of various AAVs harboring artificial gene therapies for gene silencing, such as, but not limited28335 / 708172024-081 -02 to, DNA encoding miRNA, or DNA encoding other inhibitory RNAs (RNAi), for gene silencing.

[0041] To develop a potency assay for an AAV gene therapy using miRNA-triggered RNAi, two initial obstacles were addressed. The first and most common problem when developing an in vitro potency assay for AAV is selecting a cell line permissible to transduction by the desired serotype. While AAVs exhibit remarkable tropism in vivo, they often do not transduce cells efficiently in vitro. In addition, certain tissue or cell types may not be easily cultured in vitro, necessitating the use of a proxy. The second obstacle is devising a method to quantify gene knockdown mediated by the therapeutic AAV and miRNA. Traditional methods include PCR to detect the target messenger RNA, or western blot and ELISA assays to measure protein concentration. However, these methods require extensive, time-consuming optimization.

[0042] To overcome both hurdles, a stable cell line was developed. The stable cell line, in various aspects, is made by any type of cell or “host cell” used in the field of gene therapy. Thus, the stable cell line is any cell line or any host cell line that can be transduced and stably integrate a genome. In some aspects, the cell line is any cell line that can be transduced and stably integrate a lentiviral genome. In various aspects, the cell line is made from any of a human embryonic kidney (HEK) cell, a human alveolar basal epithelial cell (A549), a HeLa cell, a Chinese hamster ovary (CHO) cell, or a myoblast cell (C2C12). In aspects of the disclosure, the stable cell line is made from a HEK cell. In some specific aspects of the disclosure, the HEK cell is a HEK293T cell.

[0043] In further aspects of the disclosure, the stable cell line is made to comprise two critical components: 1) the essential AAV receptor (AAVR) to improve AAV transduction efficiency; and 2) the target gene or disease gene of interest, i.e., the gene desired to be silenced or the gene being targeted by the inhibitory RNA (e.g., miRNA or RNAi), tagged with a reporter gene. The AAVR is encoded by the KIAA0319L gene (KIAA0319L Gene; HGNC: 30071 NCBI Gene: 79932; Ensembl: ENSG00000142687; OMIM®: 613535; UniProtKB / Swiss-Prot: Q8IZA0)). Thus, the KIAA0319L gene, or a functional fragment thereof, is used in the development of the stable cell line. A gene tagged with a reporter gene is a protein-coding gene that produces a visually identifiable characteristic when expressed in a cell.

[0044] In some aspects, the target gene, or “tagged gene”, is positioned in the gene cassette so that there is no expression of the target gene’s protein, but only expression of the reporter gene. For example, in exemplary aspects, the DNA expression cassette of the target gene is from 5’ to 3’ provided as “promoter-reporter*stop-Gol. In exemplary aspects28335 / 708172024-081 -02 disclosed herein, the DNA expression cassette of target gene is Promoter-Ren / 7 / a*stop-Gol, and, thus, only the Renilla luciferase reporter is being translated, which provides an added benefit if studying a toxic gene, such as, for example, PMP22 or DUX4.

[0045] Exemplary cell lines of the disclosure were made with two exemplary target genes, i.e., PMP22 and DUX4. The term CMT1 A cell line, as used herein, is used interchangeably or is synonymous with the term PMP22 cell line. PMP22 is the gene of interest and CMT 1 A is a disease condition that results from aberrant expression of the PMP22 gene. The term FSHD cell line, as used herein, is used interchangeably or is synonymous with the term DUX4 cell line. DUX4 is the gene of interest and FSHD is a disease condition that results from aberrant expression of the DLIX4 gene.

[0046] The PMP22 or DLIX4 open-reading frames were inserted as the 3’ UTR of rLuc, creating a fusion mRNA instead of a fusion protein. This design prevented any potential toxicity that could arise from PMP22 or DLIX4 protein expression, while also providing mRNA binding sites for the engineered miRNAs, thereby allowing quantification of PMP22 or DLIX4 gene knockdown in a dose-dependent manner following AAV treatment or using any gene silencing I gene editing modality. This approach can be adapted by replacing PMP22 or DUX4 with any gene sequence in the rLuc 3’ UTR, if the target sequence meets lentiviral packaging requirements. As such, this methodology may help expedite potency assay development to aid in regulatory approval of gene therapies targeting dominant diseases.

[0047] A key showing abbreviations used for exemplary cell lines of the disclosure for two exemplary target genes, i.e., PMP22and DUX4, are provided in the table below. This table provides a key to the stable cell line clone name in the initial experiments, i.e., the detailed name, with its equivalent reference C# name in subsequent experiments. Thus, for example, Mol 5 E5 is the same cell line as C4, as its name was simply changed in later experiments (beginning at Example 4), as described in the disclosure.28335 / 708172024-081 -02

[0048] The essential AAVR or “AAVR” serves as a critical host factor for all tested AAV serotypes. AAVR is important for in vivo gene delivery as it is a universal receptor involved in AAV infection. AAVR is a protein capable of rapidly endocytosing from the plasma membrane and trafficking to the trans-Golgi network. In some aspects, the AAVR gene is any AAVR gene or a variant thereof comprising AAVR biological activity.

[0049] In some aspects, the AAVR gene is introduced into the cell line via a lentiviral vector. In some more specific aspects, the AAVR gene is introduced into the cell using pLenti-CMV-Puro-AAVR-FLAG (Plasmid #166716 (Addgene)). Plasmid #166716 comprises the entire sequence used for transducing the AAVR component of a host cell of the disclosure. In some aspects, therefore, Plasmid #166716 is used in the first vector, as disclosed herein, for creating the stable AAVR component of the host cell or the cell line of the disclosure. In some aspects, the AAVR gene transduced into the cell line is KIAA0319L (Pillay et al., Nature. 2016 Feb 4;530(7588):108-12. doi: 10.1038 / nature16465. Epub 2016 Jan 27). KIAA0319L is the official symbol for AAVR (Gene ID: 79932 Uniprot ID: Q8IZA0 (-1 is canonical seq)).

[0050] In some aspects, the disclosure provides a method for measuring the potency of a silencing gene therapy, wherein the method, in some aspects, comprises transducing a host cell with an AAV comprising a DNA comprising a nucleotide sequence or sequences which knock down gene expression of the target gene; measuring reporter gene expression in the host cell and a control; and determining a level of RNA inhibition based upon the difference in reporter gene expression in the host cell and the control. This method is designed to be used with both any method for silencing or knocking down gene expression and with any target gene of interest, and is not limited to the exemplary inhibitory microRNAs or the target genes described herein. Such methods for silencing or knocking down gene expression include, but are not limited to, microRNAs, RNA interference (RNAi) using small interfering28335 / 708172024-081 -02RNA (siRNA) or short hairpin RNA (shRNA), antisense oligonucleotides, morpholino oligonucleotides ("morpholinos"), ribozymes, and CRISPR-based approaches like CRISPR interference (CRISPRi). Such target gene is any gene which is targeted to be silenced or knocked down. Thus, in some aspects, such target gene is a gene that when expressed or over-expressed is associated with a particular disease or disorder.

[0051] In some aspects, the disclosure provides a method for measuring the potency of a silencing gene therapy, the method comprising transducing a host cell with an AAV comprising a DNA comprising a nucleotide sequence encoding an inhibitory RNA designed to inhibit expression of the target gene; measuring reporter gene expression in the host cell and a control; and determining a level of RNA inhibition based upon the difference in reporter gene expression in the host cell and the control. As discussed herein above, this method is designed to be used with any method for knocking down gene expression and with any target gene of interest and is not limited to the exemplary gene knockdown methodology or target genes exemplified herein.

[0052] In exemplary aspects of the disclosure, the target gene is PMP22 or DUX4. Peripheral myelin protein 22 (PMP22) is a protein that plays a vital role in the development and maintenance of myelin, a protective substance that covers nerves. PMP22 is also known as growth arrest-specific protein 3 (GAS-3). Mutations in the PMP22 gene can cause changes in the expression of PMP22, which can result in several neuropathies, including, but not limited to, Charcot-Marie-Tooth disease type 1 A (CMT1A) and Hereditary Neuropathy with liability to Pressure Palsies. DUX4, or double homeobox 4, is a transcription factor gene that plays a role in early development, the testes of adult males, and certain diseases, such as muscular dystrophy and cancer. Notably, aberrant DUX4 expression is linked to facioscapulohumeral dystrophy (FSHD) and some cancers. FSHD is a muscular dystrophy caused by DLIX4 expression in skeletal muscle. DLIX4 expression in cancer cells can activate an early embryonic program that may contribute to cancer progression and metastasis.

[0053] In some aspects, the reporter gene is any gene that encodes a protein that has a visually identifiable characteristic when expressed in a cell. In some aspects, therefore, the reporter gene is any reporter gene which is able to be easily assayed for expression of the target gene. In some aspects, the reporter gene is a gene that encodes a luciferase, a fluorescent protein (including, but not limited to, a green fluorescent protein or a red fluorescent protein), a LacZ, or a beta-lactamase. In some aspects, the reporter gene is a luciferase. Luciferase enzymes isolated from different animal species have inherent variability in light emission, allowing two or more luciferase enzymes to be used in combination for multiplex analyses, including in vivo imaging, cell viability and single and28335 / 708172024-081 -02 dual-spectral luciferase reporter assays. Additionally, luciferase reactions are classified as having either flash or glow kinetics, which have specific detection sensitivities and emission duration times to accommodate different experimental designs. In some aspects, the luciferase is Ren / 7 / a-luciferase.

[0054] In some aspects, therefore, the reporter gene is Renilla luciferase. Renilla luciferase is the reporter gene used in various exemplary aspects of the disclosure because it enables easy quantification of gene knockdown (Fig. 1 A). When the stable cell line is treated with a therapeutic AAV, transduction is improved by the presence of AAVR, the engineered miRNA will be expressed, and the Renilla luciferase-target gene transcript will be degraded, resulting in reduced Renilla luciferase signal that can be easily quantified using a Renilla luciferase assay (Fig. 1 B). In some aspects, a commercial Renilla Luciferase assay kit (Promega, i.e., E2810) is used to measure expression of the reporter.

[0055] In various aspects of the disclosure, an antibiotic resistance gene is used. An antibiotic resistance gene (ARG) is a gene that provides instructions for producing a protein that makes antibiotics ineffective. There are several thousand known ARGs that can confer resistance to almost all clinically used antibiotics. Thus, the products and methods of the disclosure are not limited to specific ARGs but instead may be practiced with any ARG known in the art. In some aspects, an antibiotic resistance gene of the disclosure, is a neoymycin, a puromycin, a blasticidin, a hygromycin, a mycophenolic acid, or a zeocin resistance gene. In some aspects, only one vector comprises an antibiotic resistance gene. In some aspects, the first vector comprises one antibiotic-resistance gene and the second vector comprises the same antibiotic resistance gene. In some aspects, the first vector comprises one antibiotic-resistance gene and the second vector comprises a different antibiotic resistance gene. For example, in various aspects, the first vector comprises a puromycin resistance gene, and the second vector comprises a neomycin or blasticidin resistance gene.

[0056] An antibiotic resistance gene is used, in various aspects, for cell selection. Thus, cells that express the antibiotic resistance gene, i.e., cells that were successfully transduced, are selected by their ability to grow in the presence of antibiotic in the media. For example, cells transduced successfully with the neomycin resistance gene “NeoR” are able to in media comprising geneticin (e.g., G418).

[0057] In various aspects of the disclosure, a promoter or enhancer is used to regulate expression of the target gene. In various aspects, the promoter or enhancer may be a ubiquitous promoter or enhancer. In some aspects, a promoter that targets the gene of interest to certain cell types is used. In some aspects, the promoter is any of U6, U7, tRNA,28335 / 708172024-081 -02H1 , CMV, minimal CMV, T7, EF1 -alpha, Minimal EF1 -alpha, or a tissue-specific promoter including, but not limited to, a muscle-specific promoter, a neurospecific promoter, a Schwann cell-specific or myelin-specific promoter, or a cardiac-specific promoter. In some aspects, the promoter is CMV, U6 or H1 . In some aspects, the muscle-specific promoter is unc45b, muscle creatine kinase (MCK), tMCK, minimal MCK, CK6, CK7, CK8, alpha-myosin heavy chain enhancer- / MCK enhancer-promoter (MHCK7), or CK1 . In some aspects, the myelin-specific promoter is myelin specific protein-Z (MPZ, P0), peripheral myelin protein, 22 (PMP22), myelin basic protein (MBP), or SRY (sex determining region Y)-box 10 protein (SOX10). In some aspects, the cardiac-specific promoter is alpha-myosin heavy chain enhancer- / MCK enhancer-promoter (MHCK7), the 250-bp fragment of the myosin light chain-2v (MLC-2v) gene promoter (MLC250), cardiac troponin T (cTnT) promoter, the a- myosin heavy chain (a-MHC) promoter, muscle creatine kinase (MCK), tMCK, minimal MCK, CK6, CK7, CK8, or CK1.

[0058] In one aspect of the disclosure, therefore, a stable cell line is generated. The pipeline for stable cell line generation is demonstrated in the schematic shown in Fig. 2. As shown in Fig. 2, two lentiviruses need to be produced, one harboring a flag tagged AAVR (AAVR.Flag) with a first antibiotic-resistance gene, e.g., a puromycin resistance gene, and the other containing the Renilla luciferase-tagged target gene with a second antibioticresistance gene, e.g., either a neomycin or blasticidin resistance gene.

[0059] In an aspect of the disclosure, the stable cell line is used to measure potency. Thus, the disclosure further provides a method for measuring the potency of a silencing gene therapy, the method comprising: transducing the host cell or stable cell line as disclosed herein above with an AAV comprising a DNA comprising a nucleotide sequence encoding an inhibitory RNA designed to inhibit expression of the target gene; measuring reporter gene expression in the host cell and a control; and determining a level of RNA inhibition based upon the difference in reporter gene expression in the host cell and the control.

[0060] The methods of the disclosure comprise using any control reasonable to one skilled in the art. In some aspects, the control is an untreated stable cell line or the stable cell line transfected with an empty capsid from the same serotype, or containing a scrambled cassette, or containing a miRNA (also referred to herein as miR) that does not target the target gene. In exemplary aspects, the control is an untreated stable cell line.

[0061] In some aspects, a comparison is made between the maximum relative light units, “max RLU” (100%) signal from control cells with no AAV treatment (RLU No Trx) versus cells treated with an AAV comprising a DNA encoding an inhibitory RNA designed to inhibit28335 / 708172024-081 -02 expression of the target gene (RLU Trx). Thus, a comparison is made between relative light units measured in treated versus untreated (RLU Trx / No Trx) cells.

[0062] In some aspects, the control is a stable cell line transduced with an empty capsid from the same serotype, or containing a scrambled cassette, or containing a miR that does not target the target gene.

[0063] In some aspects, the level of decrease in reporter signal, as an indicator of the level of knockdown, is measured as percent RLU compared to control. Thus, in some aspects, the level of knockdown is at least or about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. In some aspects, the level of decrease, or the amount of knockdown, is modulated by increasing or decreasing the multiplicity of infections (Mols).

[0064] In some aspects, therefore, the host cell, cell line, and methods of the disclosure are used in the field of gene therapy using a silencing gene therapy. In some aspects, such silencing gene therapy utilizes RNA interference. RNA interference (RNAi) or post- transcriptional gene silencing (PTGS) is a conserved biological response to double-stranded RNA that mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes. Some of the main types of RNAi include, but are not limited to microRNA (miRNA), small interfering RNA (siRNA), and short hairpin RNA. miRNAs are a more general suppressive tool, formed from singlestranded RNA precursors and characterized by their distinctive hairpin shape. siRNAs are highly specific and usually synthesized to reduce the translation of specific messenger RNAs (mRNAs). shRNAs are sequences of RNA, typically about 80 base pairs in length, that include a region of internal hybridization that creates a hairpin structure. Regardless of type of RNAi, RNA interference works in approximately the same way. These small RNA molecules connect to and activate protein complexes, most notably the RNA-induced silencing complex (RISC). Once bound, they can bind to their target mRNAs and both physically prevent ribosomes from continuing to synthesize the associated protein and mark that mRNA for destruction.

[0065] In some aspects, RNAi refers to post-transcriptional control of gene expression mediated by miRNAs. The miRNAs are small (21-25 nucleotides), noncoding RNAs that share sequence homology and base-pair with 3' untranslated regions of cognate messenger RNAs (mRNAs). The interaction between the miRNAs and mRNAs directs cellular gene silencing machinery to prevent the translation of the mRNAs. The RNAi pathway is28335 / 708172024-081 -02 summarized in Duan (Ed.), Section 7.3 of Chapter 7 in Muscle Gene Therapy, Springer Science + Business Media, LLC (2010).

[0066] As an understanding of natural RNAi pathways has developed, researchers have designed artificial miRNAs for use in regulating expression of target genes for treating disease. As described in Section 7.4 of Duan, supra, artificial miRNAs can be transcribed from DNA expression cassettes. The miRNA sequence specific for a target gene is transcribed along with sequences required to direct processing of the miRNA in a cell. Viral vectors, such as adeno-associated virus (AAV) have been used to deliver miRNAs to muscle [Fechner et al., J. Mol. Med., 86: 987-997 (2008)].

[0067] In some aspects, the products and methods of the disclosure use AAV as a vector. AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic. Moreover, AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo. Moreover, AAV transduces slowly dividing and non-dividing cells, and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is infectious as cloned DNA in plasmids which makes construction of recombinant genomes feasible. Furthermore, because the signals directing AAV replication, genome encapsidation and integration are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA. The rep and cap proteins may be provided in trans. Another significant feature of AAV is that it is an extremely stable and hardy virus. It easily withstands the conditions used to inactivate adenovirus (56o to 65oC for several hours), making cold preservation of AAV less critical. AAV may even be lyophilized. Finally, AAV- infected cells are not resistant to superinfection.

[0068] In various aspects, such AAV is any AAV known in the art. In some aspects, the AAV lacks rep and cap genes. In some aspects, the AAV is a recombinant AAV (rAAV) or a self-complementary recombinant AAV (scAAV). In various aspects, the AAV is AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh74, AAV.rh8, AAV.rhW, AAV11 , AAV12, AAV13, AAV-anc80, AAV-B1 , AAV-BR1 , AAV.PHP.EB, AAVv66, AAV2 / 1 , AAV2 / 8, or AAV2 / 9, AAVMYO, MYOAAV, MYOAAV1A, MYOAAV2A, MYOAAV3A, modified AAV9 (mAAV9), or AAV-SLB101 , or any derivative thereof, or any modified AAV, chimeric AAV, or variant thereof.28335 / 708172024-081 -02

[0069] This entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features are not found together in the same sentence, or paragraph, or section of this document. The disclosure also includes, for instance, all embodiments of the disclosure narrower in scope in any way than the variations specifically mentioned above. With respect to aspects of the disclosure described as a genus, all individual species are considered separate aspects of the disclosure. With respect to aspects of the disclosure described or claimed with "a" or "an," it should be understood that these terms mean "one or more" unless context unambiguously requires a more restricted meaning.

[0070] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the disclosure.

[0071] The term "and / or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term."

[0072] The term "about" or "approximately" as used herein means within 20%, preferably within 10%, more preferably within 5%, and even more preferably within 2.5% of a given value or range. It includes, however, also the concrete number, e.g., about 10 includes 10.

[0073] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or step. When used herein the term "comprising" can be substituted with the term "containing" or "including" or sometimes when used herein with the term "having."

[0074] When used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0075] In each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms.

[0076] It should be understood that this disclosure is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular28335 / 708172024-081 -02 embodiments or aspects only and is not intended to limit the scope of the subject matter of the disclosure, which is defined solely by the claims.

[0077] The practice of a method disclosed herein, and individual steps thereof, can be performed manually and / or with the aid of or automation provided by electronic equipment. Although processes have been described with reference to particular embodiments, a person of ordinary skill in the art will readily appreciate that other ways of performing the acts associated with the methods may be used. For example, the order of various of the steps may be changed without departing from the scope or spirit of the method, unless described otherwise. In addition, some of the individual steps can be combined, omitted, or further subdivided into additional steps.

[0078] All publications and patents cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[0079] A better understanding of the disclosure and of its advantages will be obtained from the following examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the disclosure. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.EXAMPLES

[0080] Additional aspects and details of the disclosure will be apparent from the following examples, which are intended to be illustrative rather than limiting.Example 1Materials and MethodsGeneration of proviral lentiviral plasmids and lentiviral vector production

[0081] The AAVR.flag lentiviral plasmid was acquired from Addgene (#166716) and used to generate lentiviral vectors (LVs). To generate C4CMT1Acells, GFP was replaced in the pLenti-CMV-GFP-Neo plasmid (Addgene, #17447) with rLucPMP22using Xbal and Sall restriction enzyme sites. The rLucPMP22cDNA was first generated by inserting the full-length human PMP22 open reading frame and 3’ UTR within the psiCHECK2 dual luciferase vector (Promega). This construct was inserted after the rLuc stop codon, thereby yielding a fusion28335 / 708172024-081 -02 mRNA in which PMP22 serves as the rLuc 3’ UTR. Then, rLucPMP22DNA was PCR amplified to add 5’ Avril and 3’ Sall restriction sites and ligated into the same sites on the pLenti backbone using DNA ligation (Takara; #6023) per manufacturer’s instructions. To generate raw materials to create FSHD cell lines, a PCR-amplified rLucDUX4cDNA containing 5’ Sall and 3’ Xbal restriction sites was inserted and this fragment was cloned into pLenti-CMV- Blast-empty (Addgene, #17468) digested with the same enzymes. rLucDUX4was PCR- amplified from a previously described plasmid (Saad et al., Nat Commun. 2021 ; 12(1 ):7128.). The pLenti-rLucPMP22-Neo and pLenti-rLucDUX4-Blast plasmids were sequence-confirmed prior to lentiviral production. All three lentiviral vectors were made by Genewiz from Azenta Life Sciences.Stable HEK293T cell production and clonal isolation

[0082] Three VSVG-pseudotyped lentiviral vectors were generated and carried the following constructs: (1 ) flag-tagged AAVR (AAVR.flag) co-expressing a puromycin resistance (PuroR) gene; (2) rLucPMP22co-expressing a neomycin resistance (NeoR) gene; and (3) rLucDUX4co-expressing a blasticidin resistance gene (BlastR). Stable HEK293T cells were generated using dual lentiviral transduction, with one vector expressing AAVR.flag and a second expressing gene-relevant luciferase reporters (rLucPMP22for CMT1A cells, or AAVR.flag and rLucDUX4for FSHD cells). The peripheral myelin protein 22 (PMP22) gene and protein are referenced as NM 000304.4 (GenBank) and Q01453 (Uniprot). The DUX4 gene and protein are referenced as NG_203331.1 (GenBank) and Q9UBX2 (Uniprot). The KIAA0319-LIKE; KIAA0319L (AAVR) gene and protein are referenced as NM 024874.5 (GenBank) and Q81ZA0 (Uniprot). HEK293T cells were co-transduced with the described vectors at MOI of 5, 2.5, 1.25, 0.625, or 0.325, with an additional untransduced well serving as a selection indicator. Each LV was diluted in 500 pl of Dulbecco’s Modified Eagle Medium (DMEM) + l Omg / mL polybrene to the indicated MOI and added to individual wells on a 6- well plate. A reverse transduction was conducted by adding 1 mL of DMEM+10mg / mL of polybrene containing 50,000 HEK293T cells to each well. The 6-well plate was incubated at 37QC, 5% CO2 for 72hrs prior to adding selection antibiotics. CMT1A / PMP22 cell lines were selected using puromycin (2 pg / mL) and G418 (600 pg / mL). FSHD / DUX4 cell lines were selected using puromycin (0.5 pg / mL) and blasticidin (2.5 pg / mL). Media was replaced every 48-72 hours until all cells in the control well were dead and transduced cells reached 50-60% confluency. Transduced cells were transferred to a 10cm plate for further expansion and single cell isolation.28335 / 708172024-081 -02Single cell isolation

[0083] Transduced cells were diluted to a concentration of 5 cells / mL in a 50 / 50 mix of 0.22 pm filtered conditioned media (CM) from untransduced HEK293T cells and DMEM + 1% FBS. 100 pl of diluted cells were seeded to each well of a 96-well plate. Plates were then incubated undisturbed for one week at 37 -C, 5% CO2. Wells with surviving single colonies were expanded to 20-40% confluency, then transferred to a 24-well plate. After clones reached 50-60% confluency on a 24-well plate, they were transferred to a 6-well plate, until once again reaching 50-60% confluency, at which point they were transferred to a 10 cm plate. The 10 cm plate was then used for further experiments, passaging, and generating a liquid nitrogen stock.Renilla luciferase assay

[0084] To detect Renilla luciferase signal in the stable cell line clones, 40,000 cells were plated in triplicate wells on a 96-well plate. A Renilla luciferase assay kit (Promega catalog number E2820) was then used per manufacturer’s instructions. In short, 2-4 hours after cells attached to the plate, growth media was removed and replaced with 1x lysis buffer from the Promega kit and incubated with gentle agitation for 20-30 minutes. After lysis, the 96-well plate was placed in a GloMax plate reader. The GloMax injector dispense 100 pl of 1x Renilla substrate per well followed by a 2 second hold and 10 second integration, reported in relative light units (RLU). The RLU of each well was then graphed using GraphPad Prism 10.Anti-flag western blot

[0085] 5 million GMT 1 A or FSHD stable cells were pelleted at 500 xg for 5 minutes and lysed with 1 mL ice-cold RIPA buffer (Thermo Scientific, catalog #89901 ) for 30 minutes. Lysates were centrifuged at 16,000 xg for 10 minutes. 5pl was then added to reducing Laemmli SDS sample buffer with 2-betamercaptoethanol (BME) and run on a 4-20% Mini- PROTEAN® TGXTMprecast protein gel (BioRad, Catalog number 4561094). Samples were transferred onto a PVDF membrane using the Trans-Blot turbo transfer system and RTA transfer kit (Biorad, Catalog number 1704274). Membranes were then blocked using 1% dehydrated milk in TBS-T and treated with horseradish peroxidase (HRP)-coupled anti-flag (a-Flag.HRP) antibody (Proteintech, catalog number HRP-66008) at a 1 :15,000 dilution. Each blot was then washed and imaged with Immobilon™ western chemiluminescent HRP substrate (ECL Cat: WBKLS0500) using a ChemiDoc-MP by BioRad.28335 / 708172024-081 -02AAV production

[0086] All AAV vectors used in this study were produced by Andelyn Biosciences (Columbus, Ohio), except SLB101 ,mi405-No T6, which was produced at the University of Massachusetts viral vector core (Worcester, Massachusetts).AAV.GFP transduction imaging

[0087] 10,000 unmodified or stable HEK293T cells were seeded on a 96-well plate and allowed to adhere for 2-4 hrs. Cells were then transduced with 5 different AAV serotypes containing a CMV-GFP genome: AAV9, AAV6, MYOAAV3A, and MYOAAV2A (MOI 2.67e6) and SLB101 (MOI 1 .6e6). 24hrs later, wells were qualitatively assessed using fluorescent microscopy. To quantify GFP expression at 48hrs, media was replaced with 50 pl PBS and total green fluorescence measured using a Promega GlowMax instrument. RFU was normalized to unmodified HEK293T cells and percent RFU (%RFU) plotted using GraphPad Prism 10.Potency assay using Renilla luciferase as outcome measure

[0088] To test the potency of AAV vectors, 10,000 cells of each stable cell line were seeded on a 96-well plate. Following a 2-hr adherence period, cells were treated with therapeutic AAV vectors at an MOI of 9e5 (low dose) or 8e6 (high dose) for CMT1A cell lines, and MOI of 1 .6e6 (low dose) and 8e6 (high dose) for FSHD cell lines. The rLuc signal was measured 24hrs later for CMT1A cell lines and 48hrs later for FSHD cell lines, using Promega’s Renilla Luciferase Assay System (Cat: E2820). In short, media was removed and replaced with 30 pl of 1 x Renilla Luciferase Assay Lysis Buffer and placed on an orbital shaker for 15-20 minutes. Lysates were then transferred to a black bottom, black walled 96 well plate (Millipore, catalog number MSSBNFX40) and placed in a GlowMax instrument. Renilla luciferase signal was measured one well at a time using the following instrument protocol parameters: First 100 pL of Renilla Luciferase Assay Reagent was added to a well followed by a 2 second hold. Luminescence was then read at an interval of 0.3 seconds for a total of 10 seconds. All measurements were then integrated and reported as a single RLU value. The process was then repeated for each well until all wells were measured. RLU was normalized to untransduced stable cells and plotted using GraphPad Prism 10.Example 2 Initial Proof of Concept Study for Potency Assay

[0089] The strategy for potency assay development as described herein required the creation of AAV-permissive stable cell lines capable of producing quantifiable and reproducible readouts of target gene knockdown using a simple reporter assay. In this initial28335 / 708172024-081 -02 study, three such cell lines - one for CMT 1 A and two for FSHD - were generated, each using similar methods.

[0090] To carry out an initial proof of concept study, a stable cell line was made using HEK293T cells and PMP22 as the target gene. When duplicated and overexpressed, PMP22 causes Charcot-Marie-Tooth disease type 1A (CMT1 A). Dual-lentivirus transduction was conducted in a 6-well plate on 50,000 HEK293T cells in the presence of 10ug / mL of polybrene with a dilution series of 5 different multiplicity of infections (Mols) of each lentivirus: 5, 2.5, 1 .25, 0.6, and 0.3. The cells were incubated for 72 hours in a 37°C, 5% CO2 incubator. After 72 hours, media was removed and replaced with fresh media containing both selection antibiotics. After all cells were dead in the control well, and the transduced wells were 20-50% confluent, the polyclonal cells were expanded into 10 cm plates without antibiotic. Once the 10 cm plates were 50-80% confluent, cells were diluted to 5 cells / mL and plated in 96-well plates for single cell isolation. Single cell colonies were confirmed using microscopy and expanded. Once single clones were sufficiently expanded, the cells were plated in a 96-well plate and tested for the presence of Renilla luciferase using a Renilla luciferase assay (Fig. 3A). In this first proof of concept study, a neomycin resistance gene was used, however, HEK293T cells already contain neomycin resistance, lowering the success rate for the presence of Renilla luciferase in the generated clones. However, one clone, Mol-5 E5, showed robust Renilla luciferase signal. Thus, this clone, i.e., Mol-5 E5, was then tested for the presence of AAVR.Flag using an anti-flag western blot (Fig. 3B). Both gene elements were confirmed in the stable cell line, i.e., Mol-5 E5.

[0091] Next, tests were carried out to confirm that the presence of AAVR improved AAV transduction over unmodified HEK293T cells. The stable cell line was treated with scAAV9- EGFP and it was found that after 24hrs, there were more green positive cells than HEK293Ts (Fig. 4A). Quantification of GFP after 48hours showed significant increase in the GFP signal at all Mols tested (Fig. 4B).

[0092] Next, experiments were carried out to confirm that the therapeutic AAV, scAAV9- mi871 , which expresses a miRNA engineered to knockdown PMP22 as treatment for Charcot-Marie-Tooth disease type 1A (CMT1 A), was able to reliably knockdown the Renilla signal. Two independent AAV batches of scAAV9-mi871 were tested, and both were able to reliably and robustly knock down Renilla signal, thus providing an easy-to-use assay to quantify gene knockdown for vector release (Fig. 4C).28335 / 708172024-081 -02Example 3 FSHD Potency Assay

[0093] Following up to initial studies described above, further, experiments were undertaken to develop a stable cell line for a facioscapulohumeral muscular dystrophy (FSHD) potency assays. In this study, DUX4 was used as the target gene, and thus the DUX4 gene was tagged with Renilla luciferase. DLIX4 is a protein which causes FSHD when it is aberrantly expressed in skeletal muscle. In this study, the neomycin resistance was replaced with a blasticidin resistance gene in the cassette of the AAV vector comprising the Renilla luciferase gene of interest lentivirus to improve selection in HEK293T cells. As discussed in Example 2 above, HEK293T cells apparently already have a neomycin resistance gene and, therefore, a different antibiotic resistance gene was inserted into the expression cassette.

[0094] Multiple cell lines that contain a Renilla luciferase-DUX4 fusion construct were generated. It has been confirmed that the Renilla luciferase-DUX4 signal can be knocked down using two different AAV serotypes, AAV9 and SLB101 , and using a known therapeutic miRNA expression cassette (U6.mi405) (Fig. 5A-B). MicroRNA 405 (mi405) is microRNA shown to be effective in reducing DLIX4 expression (see, for example, US Patent No. 9469851).

[0095] Both the proof-of-concept study, i.e., a CMT1 A / PMP gene therapy potency assay of Example 2, and the FSHD / DUX4 potency assay of Example 3, demonstrate that a stable cell line permissible to AAV transduction can be reliably produced, and such cell line is then readily available to carry out a novel and a robust method to measure gene knockdown and / or potency. The proposed approach for potency assay development provides an easy to use and easy to adapt pipeline for therapeutic AAVs harboring miRNA expression cassettes and other mechanisms for gene knockdown release testing. To date, no other published literature has proposed using a stable cell line harboring these two components for potency assay development.Example 4Production and Clonal Isolation of Additional Stable Cell Lines for Potency Assays

[0096] Further to the potency assays described herein above, additional experiments were undertaken to develop additional stable cell lines and test their efficacy in potency assays.Stable cell line clones contain AAVR.flag and rLuc signals

[0097] To create stable cell lines harboring both AAVR and rLucPMPor rLuc°UX4, 3 different lentiviral vectors were generated and dual lentiviral transduction in HEK293T cells was28335 / 708172024-081 -02 carried out. Specifically, one lentiviral vector delivered a flag-tagged AAVR cDNA (AAVR.flag) co-expressed with a puromycin resistance (PuroR) gene for selection. The other two lentiviral vectors carried rLucPMPor rLucfUX4co-expressing a neomycin resistance (NeoR) or blasticidin resistance (BlastR) gene, respectively. Cells co-transduced with AAVR and rLucPMPlentiviral vectors were selected using puromycin and G418 while those treated with AAVR and rLucP^were selected with puromycin and blasticidin. Surviving polyclonal cells were seeded on 96-well plates at a concentration of 0.5 cells / well to enable clonal isolation. Single cell clones were confirmed using microscopy and expanded to generate a cryopreserved cell bank. Clones were then tested to confirm they possessed both AAVR.flag and the rtuc-tagged target gene (Fig. 6A-D).

[0098] The MVR / rLuc '’ cell line was first created utilizing a neomycin resistance gene and G418- selection in HEK293T cells. 22 surviving PJWP / rLu(fMPG418-resistant single cell clones were identified and expanded, and each clone was then seeded (40,000 cells per clone) on a 96-well plate. Of the 22 initial isolates, only one expressed a significant amount of rLuc (clone C4) (Fig. 6A). This low percentage of luciferase-positive clones likely resulted from reduced selection, since HEK293T cells already contain a neomycin resistance gene. Importantly, in addition to expressing Renilla luciferase, an anti-flag western blot was carried out to confirm that clone C4 had robust AAVR.flag expression (Fig. 6B). Henceforth, this stable cell line for the PMP22 or CMT1 A potency assay development was referred to as Q4CMTIA

[0099] To improve the success rate for creating a stable cell line for the development of the DUX4 or FSHD potency assay, the antibiotic resistance gene was changed on the rLucfUX4lentiviral vector from NeoRto BlastRand clones were selected using blasticidin. This change improved the selection method as it yielded 10 of 21 clones expressing rLucfUX4(Fig. 6C). To reduce the number of clones for further testing, each clone was treated with an AAV9 or SLB101 vector packaged with our therapeutic miRNA 405 (mi405) targeting DUX4 to determine which clones, if any, exhibited dose-dependent knockdown of rLuc signal following treatment (Fig. 9A-D). Two clones, C4 and C6, named C4FSHDand C6FSHD, were sensitive to the AAV therapies and thus were selected for further testing (Fig. 9A-D). An anti-flag western blot confirmed AAVR.flag expression in both C4FSHDand C6FSHD(Fig. 6D).Stable AAVR-expressing cell lines improve transduction efficiency by multiple AAV serotypes

[0100] The inventors’ hypothesis that stable AAVR over-expression would improve transduction efficiency of multiple AAV serotypes was next confirmed. C4CMT1A, C4FSHD, and C6FSHDcell lines were transduced with five AAV serotypes, 2 naturally occurring (AAV9 and28335 / 708172024-081 -02AAV6) and 3 engineered (SLB101 , MYOAAV3A, and MYOAAV2A), all expressing a CMV.GFP construct (Dewan et al., Transpl Int. 2025;38:13971 ; Gao et al., J Virol. 2004;78(12):6381 -8; Rutledge et al., J Virol. 1998;72(1 ):309-19; Tabebordbar et al., Cell. 2021 ;184(19):4919-38. e22; Weinmann et al., Nat Commun. 2020;11 (1 ):5432). Transduction efficiency was first assessed 24hrs after AAV treatment using fluorescence microscopy imaging (Fig. 7A). GFP expression was then quantified using a GloMax plate reader at 48hrs (Fig. 7B).

[0101] Compared to HEK293T cells, AAV9 transduction efficiency was significantly improved for all three clones tested. Surprisingly, the three engineered serotypes, SLB101 , MYOAAV3A, and MYOAAV2A, had significantly improved transduction on C4FSHDand C6FSHDcells, but not on C4CMT1Acells. However, C4FSHDand C6FSHDwere in part selected by screening clones using the myotropic SLB101 capsid, which could explain the differences in permissibility between FSHD and CMTI A cell lines (Fig. 9A-D). In addition, AAV6 transduction was reduced in C4CMT1Acells, but significantly improved in both FSHD cell lines, although at roughly half the levels achieved by SLB101 . Importantly, the improved tropism of AAV9 for C4CMT1Aand SLB101 for both C4FSHDand C6FSHDcell lines supports important translational programs, as AAV9, in some aspects, is used to deliver U6.miR871 to CMT1A Schwann cells and SLB101 , in some aspects, is used to deliver U6.mi405 to FSHD skeletal muscle.Therapeutic AAV potency measurements using stable cell lines

[0102] An ideal assay to measure AAV potency (AAV potency assay) is robust, reproducible, and quantifiable. To assess if the cell lines described herein could be used to measure potency, each cell line was treated with indication-relevant therapeutic AAVs, or controls, and rLucPMPor rLucfUX4signal was measured 24- or 48-hours later, respectively. For the CMT1 A (or DLIX4) potency assay, C4CMT1Acells were treated with AAV9.miR871 vector at low (9e5) and high (8e6) multiplicity of infection (MOI). For the FSHD (or PMP22) potency assay, C4FSHDand C6FSHDcells were treated with low (1 ,6e6) and high (8e6) doses of SLB101 ,mi405 vector. Both vectors produced dose-dependent rLuc reductions in their respective cell lines, relative to untreated cells (Fig. 8A). Specifically, for CMT1A, the therapeutic AAV9.miR871 vector significantly reduced rLucPMPsignal in C4CMT1Acells by 57% (low dose) and 78% (high dose) (Fig. 8A). Similarly, both FSHD cell lines, C4FSHDand C6FSHD, showed nearly identical dose-dependent rLucfUX4signal reduction of -36% (low dose) and -64% (high dose).

[0103] Assay specificity was then tested using additional controls, including inactivated therapeutic vectors and an AAV expressing a non-targeting miRNA for the relevant target28335 / 708172024-081 -02 gene in each assay. To inactivate AAV9.miR871 and SLB101.mi405 vectors, each vector was subjected to 1 , 3, 5, or 10 freeze-thaw cycles from -80QC to 37QC, and separately a second set of vectors was heat-treated (boiled) at 98QC for 5 minutes. For CMT1 A (PMP22), an MOI of 8e6 was used to treat C4CMT1Acells with (A) therapeutic AAV9.miR871 ; (B) 4 different freeze-thawed samples of AAV9.miR871 ; (C) heat-inactivated AAV9.miR871 , and (D) to remain consistent with serotype, AAV9.mi405, which targets DUX4 and not the PMP22 sequences present in the rLucPMPtranscript.

[0104] Compared to untreated C4CMT1Acells, AAV9.miR871 -treated cells showed a significant 66% reduction in rLucPMPactivity. Interestingly, up to 10 freeze-thaw cycles had no impact on AAV9.miR871 potency in this assay, as freeze-thawed AAV9.miR871 samples still triggered rLucPMPknockdown to levels produced by active AAV9.miR871 (Fig. 8B). In contrast, the boiled AAV9.miR871 and the AAV9.mi405 control failed to reduce rLucPMPactivity in C4CMT1Acells. A similar set of experiments was carried out for the FSHD (DLIX4) potency assay, using an 8e6 MOI of active SLB101.mi405, freeze-thawed SLB101 ,mi405, and heat-inactivated SLB101.mi405 in C4FSHDcells. For the non-targeting control, an AAV9.miR871 vector was used; while the control serotype is different (AAV9 vs. SLB101 ), data demonstrated that AAV9 and SLB101 both efficiently transduced FSHD (PMP22) cell lines (Fig. 7B). The results in the FSHD assay were similar to those produced in the CMT1A assay (Fig. 8B-C). Specifically, neat and freeze-thawed SLB101 ,mi405 significantly reduced rLucfUX4signal to similar levels (-60%), while heat-inactivated SLB101 ,mi405 and nontargeting AAV9.miR871 had no impact on rLu(^UX4activity compared to untreated control C4FSHDcells (Fig. 8C).

[0105] Finally, to demonstrate universality of the herein described potency assay, C4FSHDcells were transduced with the same dose (8e6 MOI) of SLB101 ,mi405, a SLB101 vector containing a control mi405 lacking a T6 terminator (mi405-No T6), and 2 other previously published DL / X4-targeting miRNAs, mi333 and mil 85, each packaged in AAV6, and both of which had been stored at 4QC for several years prior to use (Fig. 8D) (Wallace et al., Mol Ther Methods Clin Dev. 2018;8:121 -30). The SLB101 ,mi405 again silenced rLucDUX4by -60% while the absence of a terminator sequence on mi405 reduced the rLucfUX4by only -10%. The other miRNA candidates, mi333 and mil 85, significantly silenced rLucfUX4by 80- 90%, demonstrating the assay could be used to assess potency of DL / X4-targeting sequences beyond mi405 (Fig. 8D).

[0106] These experiments demonstrate that a stable cell line permissible to AAV transduction can be reliably produced, and such cell line is then readily available to carry out a novel and a robust method to measure gene knockdown and / or potency. The products and method disclosed herein provide an easy to use and easy to adapt potency assay useful in28335 / 708172024-081 -02 the development and manufacturing of therapeutic AAVs harboring miRNA expression cassettes and other mechanisms for gene knockdown release testing. To date, no other published literature has proposed using a stable cell line as described herein for potency assay development.

[0107] While the present invention has been described in terms of specific embodiments, it is understood that variations and modifications will occur to those skilled in the art.Accordingly, only such limitations as appear in the claims should be placed on the invention.

Claims

28335 / 708172024-081 -02CLAIMSWhat is claimed is:

1. A host cell comprising a first vector comprising a DNA comprising a nucleotide sequence encoding an adeno-associated virus (AAV) receptor (AAVR) or a variant thereof comprising AAVR biological activity; and a second vector comprising a DNA comprising a nucleotide sequence encoding a target gene tagged with a reporter gene.

2. The host cell of claim 1 , wherein either the first vector or the second vector is a lentiviral vector.

3. The host cell of claim 1 , wherein the first vector is a lentiviral vector and the second vector is a lentiviral vector.

4. The host cell of any one of claims 1 -3, wherein the target gene is a gene desired to be silenced or knocked down.

5. The host cell of claim 4, wherein the gene desired to be silenced or knocked down is a gene in which its expression or overexpression is associated with a disease or disorder.

6. The host cell of any one of claims 1 -5, wherein the first vector and / or the second vector further comprises an antibiotic resistant gene.

7. The host cell of claim 6, wherein the antibiotic resistance gene of the first vector and the antibiotic resistance gene of the second vector are different.

8. The host cell of claim 6 or 7, wherein the antibiotic resistance gene is a puromycin resistance gene, a neomycin resistance gene, a blasticidin resistance gene, a hygromycin resistance gene, a mycophenolic acid resistance gene, or a zeocin resistance gene9. The host cell of any one of claims 6-8, wherein the antibiotic resistance gene of first vector is a puromycin resistance gene.

10. The host cell of any one of claims 6-8, wherein the antibiotic resistance gene of the second vector is a neomycin resistance gene or a blasticidin resistance gene.11 . The host cell of any one of claims 1 -10, wherein the first vector and / or the second vector further comprises a promoter.28335 / 708172024-081 -0212. The host cell of any one of claims 1 -11 , wherein the reporter gene is a gene encoding a luciferase, a fluorescent protein, a LacZ, or a beta-lactamase.

13. The host cell of any one of claims 1 -12, wherein the reporter gene is a gene encoding a luciferase.

14. The host cell of claim 13, wherein the luciferase is a Renilla luciferase.

15. The host cell of any one of claims 1 -14, wherein the cell is a human embryonic kidney (HEK) cell, a human alveolar basal epithelial cell (A549), a HeLa cell, a Chinese hamster ovary (CHO) cell, or a myoblast cell (C2C12).

16. The host cell of claim 15, wherein the HEK cell is a HEK293 cell.

17. The host cell of any one of claims 1 -16, wherein the DNA comprising the nucleotide sequence encoding the target gene tagged with the reporter gene is positioned or designed in the vector gene so that the target gene is not transcribed or translated in the cell, whereas the reporter gene tagged to the target gene is transcribed and / or translated in the cell.

18. A cell line comprising a plurality of the host cells of any one of claims 1-17, wherein the cell line is selected for its ability to grow in media comprising at least one antibiotic, wherein the at least one antibiotic is the antibiotic of the antibiotic resistance gene present in the first and / or second vector.

19. A method for measuring the potency of a silencing gene therapy, the method comprising: transducing the host cell of any one of claims 1-18 with an AAV comprising a nucleotide sequence that inhibits expression of the target gene; measuring reporter gene expression in the host cell and a control; and determining a level of RNA inhibition based upon the difference in reporter gene expression in the host cell and the control.

20. The method of claim 19, wherein the level of RNA inhibition is a measure of the potency of the silencing gene therapy.21 . The method of claim 19 or 20, wherein the nucleotide sequence that inhibits expression of the target gene is a nucleotide sequence encoding a microRNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense oligonucleotide, morpholino oligonucleotide, ribozyme, or CRISPR interference (CRISPRi) components.28335 / 708172024-081 -0222. The method of any one of claims 19-21 , wherein the nucleotide sequence that inhibits expression of the target gene is a microRNA (miRNA), a short inhibitory RNA (siRNA), or a short hairpin RNA (shRNA).

23. The method of claim 22, wherein the nucleotide sequence that inhibits expression of the target gene is a nucleotide sequence encoding a microRNA.

24. The method of any one of claims 19-23, wherein the nucleotide sequence that inhibits expression of the target gene is a nucleotide sequence that targets DUX4 or PMP22.

25. The method of claim 23, wherein the microRNA is a DLIX4 or a PMP22 microRNA and / or the target gene is DUX4 or PMP22.

26. The method of any one of claims 19-25, wherein the method is used as a potency assay to measure the potency of the AAV gene therapy product and / or measure the strength and stability of a therapeutic AAV product over time and under different storage conditions.

27. The method of any one of claims 19-26, wherein the AAV is AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh74, AAV.rh8, AAV.rhW, AAV11 , AAV12, AAV13, AAV-anc80, AAV-B1 , AAV-BR1 , AAV.PHP.EB, AAVv66, AAV2 / 1 , AAV2 / 8, or AAV2 / 9, AAVMYO, MYOAAV, MY0AAV1 A, MY0AAV2A, MY0AAV3A, modified AAV9 (mAAV9), or AAV-SLB101 , or any derivative thereof, or any modified AAV, chimeric AAV, or variant thereof.

Images