VIRAL VECTOR POTENCY ASSAY

Cross Reference To Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63/116,472 filed November 20, 2020, the entire contents of which is hereby incorporated by reference.

Background

[0002] Viral vector mediated gene therapy is a rapidly developing therapeutic field. Many aspects of therapeutic viral vectors will need to be evaluated to determine safety and efficacy prior to use as a therapy.

Summary

[0003] The present disclosure recognizes the need to effectively and accurately detect the potency of gene therapy vectors. Understanding the potency of a gene therapy vector is an important aspect to understanding the efficacy of the vector as a therapy, as well as the potential doses required to achieve a safe, yet therapeutic effect. Potency of viral vectors can be determined by evaluating the biological activity of the viral vector. Biological activity can be determined by evaluating the level of expression and/or the function/activity of the pay load, e.g., protein expressed in cells in cells. Accordingly, provided herein are methods for determining the level of expression and activity (e.g., functional potency) of protein expressed by a gene therapy vector, e.g., a REP1 vector (e.g., human REP1 vector).

[0004] In some embodiments the present disclosure provides a method of determining the biological activity of a protein encoded by a recombinant viral vector comprising the steps of: (a) transducing HEK293T cells with the recombinant viral vector comprising a nucleic acid encoding a protein; (b) culturing the transduced cells; (c) lysing the transduced cells to form a lysate; (d) diluting the lysate; and (e) performing a prenylation assay on the lysate, thereby determining the biological activity of the protein encoded by the recombinant viral vector. [0005] In some embodiments the present disclosure provides a method of determining the quantity of a protein expressed comprising the steps of: a) transducing cells with a recombinant viral vector comprising a nucleic acid encoding the protein; b) culturing the transduced cells a sufficient amount of time to allow for expression of the protein; c) lysing the transduced cells; and d) detecting the quantity of the protein expressed. In some embodiments, the protein is REP1. In some embodiments, the cells are HEK293T.

[0006] In some embodiments, the present disclosure provides A method of determining the biological activity of a protein encoded by a recombinant viral vector comprising the steps of: (a) transducing HEK293T cells with the recombinant viral vector comprising a nucleic acid encoding a protein; (b) culturing the transduced cells; (c) lysing the transduced cells to form a lysate; (d) diluting the lysate; and (e) performing a prenylation assay on the lysate, thereby determining the biological activity of the protein encoded by the recombinant viral vector. In some embodiments, the protein is REP1. In some embodiments, the protein is human REP1.

Brief Description of the Drawing

[0007] Figures 1A-1B show Rep1 expression in AAV-infected HEK and Huh7 cells. (A) HEK293 and HEK293T cells were infected at 2.5E5 vg/cell (titer determined by qPCR) for 3 days and lysate was blotted for Rep1 and Actin. (B) HEK293T and Huh7 cells were infected at 5 different MOIs for 3 days and Rep1 expression was measured by MSD.

[0008] Figure 2 shows morphology of HEK293T cells after rAAV2-Rep1 infection. HEK293T cells were infected by rAAV2-Rep1 at various MOIs and observed 64 hours after infection.

[0009] Figure 3 shows a comparison of lysis buffers. 3 days after rAAV2-Rep1 infection, cells were lysed by RIPA buffer and 3 different NETN buffers. Rep1 was detected by MSD.

[0010] Figure 4 shows Rep1 antibody concentrations for ELISA. Three concentrations for both capture and detection antibodies were tested for Rep1 ELISA (9 conditions in total). Test samples were four HEK293T samples infected at different MOIs.

Data shown were ELISA signal at 450nm adjusted by signal at 540nm.

[0011] Figures 5A-5B show Sample dilution for ELISA. Lysates of HEK293T cells infected at different MOIs were diluted in assay buffer prior to ELISA analysis. The curve shape, signal and background levels were compared to determine the optimal sample dilution factor.

[0012] Figures 6A-6B show an exemplary Meso Scale Discovery (MSD) sandwich ELISA. (A) An electrical signal passes through the bottom of each well, causing an electrochemiluminescent signal from the bound labeled antibody. (B) Reagents include two antibodies that recognize the protein of interest, and a secondary antibody linked to a Sulfo- tag. Reference: www.mesoscale.com

[0013] Figure 7 shows a comparison of MSD and ELISA. HEK293T cells were infected by rAAV2-Rep1 from 2.5E5 to 1.1E2 vg/cell. Cell lysate was tested side by side with ELISA or MSD.

[0014] Figure 8 shows curve fitting models in PLA3.0. Different infection schemes were tested to allow data to be analyzed by linear or 4PL fit. rAAV2-Rep1 was tested at 100% (standard) as well as 50% and 150% strength to assess accuracy. The RP measured was shown on the upper left corner. The high/low signal of standard curve were calculated.

[0015] Figure 9 demonstrates REP1 protein expression in HEK293 and HEK293T cells following treatment with AAV2-hREP1. HEK293 and HEK293T cells were plated at 4E+4 cells/well and infected for 3 days with 2.5E+5 VG/cell (qPCR) of rAAV2-hREP1. Cells were lysed in NETN buffer and REP1 expression was determined by Western blot with an ImageQuant LAS-4000 Luminescent Image Analyzer.

[0016] Figure 10 demonstrates REP1 protein expression in HEK293T and Huh7 cells following treatment with AAV2-hREP1. Cells were plated at 2E+4 cells/well and treated for 2 days, then lysed in NETN buffer and assayed for REP1 expression. MOI is calculated based on qPCR values. [0017] Figure 11 demonstrates effect of treatment time on assay performance. Thawed HEK293T cells were plated for 2 and 3 days respectively at a cell density of 4E+4 and 2E+4 cells/well and infected with 2.5E+5 VG/cell (qPCR) at a 1:2 and 1:1.5 serial dilution of rAAV2-hREP1. Cells were then lysed, and then assayed for potency. The quantification of prenylated Rab6a was determined by MSD immunoassay using a MSD plate reader.

[0018] Figures 12A-12B demonstrate the effect of cell density on assay performance. Figure 12A shows the effect of cell density on total protein measured in lysate. Cells were lysed in 50 μL of prenylation buffer (six replicates of each density), then total protein was measured with a Bradford assay. Figure 4B shows the effect of cell density on prenylation. HEK293T cells were plated at a density of 2E+4, 3E+4, and 4E+4 cells/well and infected with 1.25E+5 and 7.81E+3 VG/cell (qPCR) of rAAV2-hREP1. Cells were treated for 2 days, then lysed and evaluated for REP1 activity by prenylation assay.

[0019] Figures 13A-13C demonstrate the effect of viral vector dilution on assay performance. Figure 13A shows different serial dilution schemes of rAAV2-REP1. Thawed HEK293T cells were plated at a density of 4E+4 cells/well and infected with a top MOI of 2.5E+5 VG/cell (qPCR) rAAV2-hREP1. Two 10-point dilution scheme serially diluted at (A) 1: 1.5, (B) 1 :2 and (C) a third dilution scheme of 5-points at a 1 :2 dilution followed by 5-points at a 1 :3 dilution was used. The potency assay was performed and quantification of prenylated Rab6a was determined by MSD immunoassay. Figure 13B shows linear fit of the dilution scheme. The first five points of the curves described in Figure 13 A, dilutions (A) 1: 1.5, (B) 1 :2 were plotted on a double-log transformed axis and both portions are a linear fit with a respective R ² of 0.9953 and 0.9926. Figure 13C shows 5-point linear fit dilution schemes. With a 2x dilution scheme starting at 5E4 VG/cell, the 150% sample showed non- parallelism (slope ratio: 0.87). With a 2x dilution scheme starting at 2.5E4 VG/cell, the 50% sample demonstrated non-parallelism (slope ratio: 0.90). By decreasing the dilution factor and starting at 3E4 VG/cell both the 50% and 150% samples were parallel to the control (slope ratios: 0.92 and 1.00, respectively).

[0020] Figures 14A-14C show the effect of detergent on Rab6a prenylation. The prenylation reaction was performed in prenylation buffer supplemented with (A) TritonX- 100 or (B) NP-40 at a 0, 0.1, 0.25, 0.5 or 1% final concentration. The reaction was performed for 2 hours at 37°C with 20 μM GDP, 5 μM B-GPP, 2 μM GGTase-II, 30 nM hREP1and 4 μM Rab6a. Following incubation, the whole reaction was analyzed by SDS- PAGE and blotted with REP 1 antibody overnight at 4°C and streptavidin-HRP (Rab6a) for 30 minutes at RT. REP1 only (30 nM) and biotin-Rab6a (B-Rab6a, 500 ng) were loaded as positive control. LB.:immunoblot. Relative band intensity is compared in (C).

[0021] Figures 15A-15C show the effect of the freeze/ thaw cycle on HEK293T cells lysis. Thawed HEK293T cells were plated at a density of 2E+4 cells/well for 2 days. Cell culture media was removed from the wells and prenylation buffer supplemented with protease inhibitor cocktail was added to the cells. (A) Cells were frozen at -70°C for 10 minutes, then thawed on the Micromix shaker for 15 minutes (program 20, amplitude 5). (B) The process was repeated once more. (C) After the second freeze/thaw cycle, the cells were pipetted up and down 5 times.

[0022] Figures 16A and 16B shows the effect of freeze/thaw cycling lysis on protein extraction and prenylation assay. Figure 16A shows protein quantitation of HEK293T cells after lysis by NETN incubation or freeze/thaw cycling. After treatment with NETN or freeze/thaw cycling, lysates were pipetted up and down to homogenize, then centrifuged at 2200 x g for 10 minutes at 4 °C. Six replicates of lysate were used to assess protein concentration with the PierceTM BCA Protein Assay Kit (Thermo Scientific cat. # 23227). Figure 16B shows prenylation efficiency after freeze/thaw cycling. HEK293T cells were treated with 5E+4 VG/cell (ddPCR) of AAV2-hREP1 for two days, then lysed with 1 or 2 freeze/thaw cycles. Lysates were used in the prenylation reaction and prenylated Rab6a was measured by MSD.

[0023] Figure 17 shows the effect of shaking during lysis. Cells were treated with various concentrations of AAV2-hREP1 for two days, then lysed in 50 μL of prenylation buffer. MOI is calculated with qPCR titer values. During the two 20-minute thaw cycles, plate was placed on the Micromix shaker (Function 20, Amplitude 5), a standard benchtop shaker (400 RPM), or on a stationary benchtop. [0024] Figure 18 shows the effect of lysate centrifugation on prenylation signal. Cells were treated with AAV2-hREP1, then cells were freeze/thawed twice in 50 μL of prenylation buffer. Lysate was used directly in the prenylation reaction (- centrifuge) or centrifuged for 10 minutes at 2200 xg at 4 °C (+ centrifuge).

[0025] Figure 19 shows optimization of lysis buffer volume. HEK293T cells were plated at a density of 4E+4 cells/well and infected with 6.25E+4 or 7.81E+3 VG/cell of rAAV2-hREP1 (qPCR). Cells were treated for 2 days, then lysed in 50 or 80 μL of prenylation buffer. A potency assay was performed as described herein with 4 μM recombinant Rab6a and 2 μM GGTase-II. The quantitation of prenylated Rab6a was determined by MSD immunoassay.

[0026] Figure 20 shows the effect of the total protein concentration on the assay. Thawed HEK293T cells were plated for 3 days at 2E+4 cells/well and infected with 2.5E+5 VG/cell (qPCR) of rAAV2-hREP1 serially diluted at 1:2 serial dilution. Cells were lysed and the total amount of protein quantified by a BCA assay. Following cell lysis, the prenylation reaction was performed with 4.5 and 9 μg of total protein. The quantification of prenylated Rab6a was determined by MSD immunoassay using the MSD plate reader.

[0027] Figures 21A and 21B show the effect of concentration of reagents in the prenylation reaction. Figure 21 A shows the effect of the GGTase-II concentration on the prenylation reaction. Different concentration of GGTase-11 (1, 2 and 4 μM) were tested in the prenylation reaction with HEK293T cell lysate infected at 1.67E+5 or 4.95E+4 VG/cell (ddPCR). The reaction and the immunodetection was performed with Rab6a used at a final concentration of 4 μM. Figure 21B shows the effect of the Rab6a concentration on the prenylation reaction. Different concentrations of Rab6a (2, 4 and 8 μM) produced by Abeam or Jena Bioscience were tested in the prenylation reaction with HEK293T cell lysate infected at 8.85E+4 VG/cell (ddPCR). The Western blot image corroborates the MSD results, showing the greatest biotinylation at 2 μM.

[0028] Figures 22A and 22B show the effect of the length of the prenylation reaction. Thawed HEK293T cells were plated for 2 days at 4E+4 cells/well and infected with rAAV2-hREP1. Cells were then lysed, and the prenylation reaction performed at (A) 1.56E+4 and 1.25E+5 VG/cell or (B) at the highest MOI (2.5E+5 VG/cell) ( qPCR). The reaction was tested for (A) 30 minutes, 1 and 2 hours under agitation or (B) for 1.5 and 2 hours with/without agitation at 37°C.

[0029] Figure 23 shows the effect of a freezing step post-prenylation reaction.

Thawed HEK293T cells were plated for 2 days at 4E+4 cells/well and uninfected or infected with a 3.0E+4 VG/cell (qPCR) of rAAV2-hREP1. Cells were then lysed, and the prenylation reaction was performed. One week later, the samples were thawed and run side-by-side.

[0030] Figure 24 shows the effect of B-GPP on the ELISA assay format. Different concentrations of B-GPP (0, 2 and 5 mg/mL) were added to purified biotin-Rab6a serially diluted at 1:2.5 from 3000 to 77ng/mL. The mix of B-GPP and biotin-Rab6a was incubated on an ELISA streptavidin-coated plate for 1 hour at 24°C, 400rpm.

[0031] Figure 25 shows cartoons of previous and final immunoassay designs. The prior assays often begin with a streptavidin-coated plate. The immunoassay to detect a lapidated RAB6A product, as described herein, sandwiches the biotinylated Rab6a between an anti-Rab6a coated plate and a streptavidin detection reagent.

[0032] Figure 26 shows results of testing specificity and sensitivity of Rab6a antibodies by Western blot. 15 μg of HEK293T whole cell lysate were analyzed by SDS- PAGE and blotted at 4°C overnight with different dilutions of Rab6 antibodies (SCBT, clones 3G3 and 38-TB, monoclonal antibodies; Abeam, Ab 95954, polyclonal antibody) diluted in PBST-3% BSA. Ab: Abeam, kDa: kiloDaltons.

[0033] Figure 27 shows RIPA buffer improves the signal-to-background ratio. MSD plates were blocked for 1 hour at 24°C and with agitation speed of 400rpm in 1% BSA/ TBS. Purified biotin-Rab6a was serially diluted at 1:2 from 500 to 125 ng/mL in TBS-T- BSA or RIPA buffer and the quantification of biotin-Rab6a was assessed by MSD immunoassay using the MSD plate reader.

[0034] Figure 28 shows the specificity of the MSD immunoassay approach. Purified biotin-Rab6a was serially diluted in prenylation buffer or in HEK293 cell lysate from 1000 to 125 ng/mL while purified biotin-RS 1 was diluted to 1000 ng/mL. Proteins were incubated on an MSD plate coated with Rab6a capture antibody for 2 hours at 24 °C, 400rpm. The quantification of biotin-Rab6a and biotin- RS 1 was determined by MSD immunoassay using the MSD plate reader. B-RS 1: biotin- RS 1; B-Rab6a: biotin-Rab6a.

[0035] Figures 29A and 29B provide microscopy images of hTERT REP-1 cells. Cells were plated at 1.5E+4 cells/well, then imaged on the same day (Figure 29 A) or three days after plating (Figure 29B).

[0036] Figures 30A and 30B shows Western blot results of hTERT REP-1 cells treated with rAAV2-hREP1. Figure 30A: Cells were plated at 1.5E+4 cells/well and treated with 2.5E+5 VG/cell (lane 1), 6.25E+4 VG/cell (lane 2), or 0 VG/cell (lane 3), then incubated for 3 days. Cells were lysed, run on SDS-PAGE, transferred to a PVDF membrane, and probed for REP1 (top) and actin (bottom). Figure 30B: Band intensity was quantified with ImageJ and normalized for actin levels.

[0037] Figure 31 shows REP1 MSD of hTERT REP-1 cells treated with rAAV2- hREP1. Cells were plated at 1.5E+4 cells/well and treated with a 7-point serial dilution of rAAV2-hREP1 (5+E5 to 3.9E+3 VG/mL, 2-fold serial dilutions), then incubated for 3 days. Cells were lysed and run in the REP1 expression assay with MSD readout.

Definitions

[0038] About: The term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0039] Amino acid: in its broadest sense, as used herein, refers to any compound and/or substance that can be incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H ₂ N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally- occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and/or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and/or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and/or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term “amino acid” may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.

[0040] Antibody: As used herein, the term “antibody” refers to a polypeptide that includes canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen. As is known in the art, intact antibodies as produced in nature are approximately 150 kD tetrameric agents comprised of two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each) that associate with each other into what is commonly referred to as a “Y-shaped” structure. Each heavy chain is comprised of at least four domains (each about 110 amino acids long)- an amino-terminal variable (VH) domain (located at the tips of the Y structure), followed by three constant domains: CH1, CH2, and the carboxy-terminal CH3 (located at the base of the Y’s stem). A short region, known as the “switch”, connects the heavy chain variable and constant regions. The “hinge” connects CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to one another in an intact antibody. Each light chain is comprised of two domains - an amino-terminal variable (VL) domain, followed by a carboxy-terminal constant (CL) domain, separated from one another by another “switch”. Intact antibody tetramers are comprised of two heavy chain-light chain dimers in which the heavy and light chains are linked to one another by a single disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and the tetramer is formed. Naturally-produced antibodies are also glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure characterized by an “immunoglobulin fold” formed from two beta sheets (e.g., 3-, 4-, or 5-stranded sheets) packed against each other in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as “complement determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4). When natural antibodies fold, the FR regions form the beta sheets that provide the structural framework for the domains, and the CDR loop regions from both the heavy and light chains are brought together in three-dimensional space so that they create a single hypervariable antigen binding site located at the tip of the Y structure. The Fc region of naturally-occurring antibodies binds to elements of the complement system, and also to receptors on effector cells, including for example effector cells that mediate cytotoxicity. As is known in the art, affinity and/or other binding attributes of Fc regions for Fc receptors can be modulated through glycosylation or other modification. In some embodiments, antibodies produced and/or utilized in accordance with the present invention include glycosylated Fc domains, including Fc domains with modified or engineered such glycosylation. For purposes of the present invention, in certain embodiments, any polypeptide or complex of polypeptides that includes sufficient immunoglobulin domain sequences as found in natural antibodies can be referred to and/or used as an “antibody”, whether such polypeptide is naturally produced (e.g., generated by an organism reacting to an antigen), or produced by recombinant engineering, chemical synthesis, or other artificial system or methodology. In some embodiments, an antibody is polyclonal; in some embodiments, an antibody is monoclonal. In some embodiments, an antibody has constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, antibody sequence elements are humanized, primatized, chimeric, etc, as is known in the art. Moreover, the term “antibody” as used herein, can refer in appropriate embodiments (unless otherwise stated or clear from context) to any of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, in some embodiments, an antibody utilized in accordance with the present invention is in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPs™ ”); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies;, Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc], or other pendant group [e.g., poly-ethylene glycol, etc.

[0041] Biological Sample. As used herein, the term “biological sample” typically refers to a sample obtained or derived from a biological source (e.g., a tissue or organism or cell culture) of interest, as described herein. In some embodiments, a source of interest comprises an organism, such as an animal or human. In some embodiments, a biological sample is or comprises biological tissue or fluid. In some embodiments, a biological sample is a cellular lysate.

[0042] Cellular lysate: As used herein, the term “cellular lysate” or “cell lysate” or “lysate” refers to a fluid containing contents of one or more disrupted cells (i.e., cells whose membrane has been disrupted). In some embodiments, a cellular lysate includes both hydrophilic and hydrophobic cellular components. In some embodiments, a cellular lysate is a lysate of one or more cells selected from the group consisting of plant cells, microbial (e.g., bacterial or fungal) cells, animal cells (e.g., mammalian cells), human cells, and combinations thereof. In some embodiments, a cellular lysate is a lysate of one or more abnormal cells, such as cells infected by a virus. In some embodiments, a cellular lysate is a crude lysate in that little or no purification is performed after disruption of the cells; in some embodiments, such a lysate is referred to as a “primary” lysate. In some embodiments, one or more isolation, dilution, or purification steps is performed on a primary lysate; however, the term “lysate” refers to a preparation that includes multiple cellular components and not to pure preparations of any individual component.

[0043] Comprising: A composition or method described herein as "comprising" one or more named elements or steps is open-ended, meaning that the named elements or steps are essential, but other elements or steps may be added within the scope of the composition or method. To avoid prolixity, it is also understood that any composition or method described as "comprising" (or which "comprises") one or more named elements or steps also describes the corresponding, more limited composition or method "consisting essentially of" (or which "consists essentially of") the same named elements or steps, meaning that the composition or method includes the named essential elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristic(s) of the composition or method. It is also understood that any composition or method described herein as "comprising" or "consisting essentially of" one or more named elements or steps also describes the corresponding, more limited, and closed-ended composition or method "consisting of" (or "consists of") the named elements or steps to the exclusion of any other unnamed element or step. In any composition or method disclosed herein, known or disclosed equivalents of any named essential element or step may be substituted for that element or step.

[0044] Determine: Many methodologies described herein include a step of “determining”. Those of ordinary skill in the art, reading the present specification, will appreciate that such “determining” can utilize or be accomplished through use of any of a variety of techniques available to those skilled in the art, including for example specific techniques explicitly referred to herein. In some embodiments, determining involves manipulation of a physical sample. In some embodiments, determining involves consideration and/or manipulation of data or information, for example utilizing a computer or other processing unit adapted to perform a relevant analysis. In some embodiments, determining involves receiving relevant information and/or materials from a source. In some embodiments, determining involves comparing one or more features of a sample or entity to a comparable reference.

[0045] Expression: As used herein, “expression” refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and/or 3’ end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein. Expression can be evaluated by determining the level, amount, or concentration, of a polynucleotide, a polypeptide or protein, e.g., at a given point in time in a given biological sample (e.g., a lysate).

[0046] Protein: As used herein, the term “protein” refers to a polypeptide (z.e. , a string of at least two amino acids linked to one another by peptide bonds). Proteins may include moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means.

Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.

[0047] Reference: As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control.

[0048] Specificity: As is known in the art, “specificity” is a measure of the ability of a particular ligand to distinguish its binding partner from other potential binding partners.

Detailed Description of Certain Embodiments

Choroideremia

[0049] Choroideremia (CHM) is a rare, X-linked recessive retinal dystrophy caused by mutations in the CHM gene, which encodes for Rab escort protein 1 (REP1).

Choroideremia leads to degeneration of the retinal pigment epithelium (RPE) and the photoreceptors of the eye. CHM is ubiquitously expressed in human cells and encodes Rab escort protein 1 (REP1). REP1 involved in the C-terminus posttranscriptional modification of one or more Rab GTPases, the largest family within the Ras-like GTPase superfamily. This modification, known as prenylation, is catalyzed by the Rab geranylgeranyl transferase (RGGT or GGT-II) and involves the covalent attachment of one or more C20 (geranylgeranyl) isoprenoid groups to a cysteine residue within a ‘prenylation motif’ of a Rab GTPase. Without wishing to be bound by any one particular theory, REP1 assists by either presenting the unprenylated Rabs to the GGT-II and/or escorting the prenylated Rabs to their destination membrane where they play a role in vesicle trafficking.

[0050] The choroideremia-like gene (CHML) encodes for Rab escort protein 2 (REP2). REP2 shares 95% of its amino acid sequence with REP1, and studies have shown that REP2 can compensate for REP1 deficiency in most cells of the body. However, REP2 is unable to fully compensate for REP1 deficiency in the eye. In choroideremia patients, the prenylation of Rah GTPases in the eye is affected, which causes cellular dysfunction and ultimately cell death.

[0051] There is therefore a need for assays to assess the potency of gene therapy vectors. In some embodiments potency of gene therapy vectors is assessed by measuring the biological activity of gene therapy vectors for the treatment of choroideremia. For example, there is a need for reliable and sensitive in vitro assays to determine the potency of rAAV2/2-REP1. For example, there is a need for reliable and sensitive in vitro assays to determine the biological activity of rAAV2/2-REP1. In some embodiments, assessing biological activity can include determining the level of expression of a protein encoded by a recombinant viral vector for the treatment of choroideremia. In some embodiments, assessing biological activity can include determining the activity (e.g. enzymatic activity) of a protein encoded by a recombinant viral vector for the treatment of choroideremia. In some embodiments, biological activity can be determined by an enzymatic assay, e.g., a cell based enzymatic assay. In some embodiments, and enzymatic assay is a prenylation reaction reproduced in vitro to test for REP1 potency.

REP1

[0052] REP1 is a component of the RAB geranylgeranyl transferase (GGTase) holoenzyme. In the dimeric holoenzyme, the REP1 component binds unprenylated Rab GTPases and presents them to the catalytic Rab GGTase subunit for the geranylgeranyl transfer reaction. Rab GTPases need to be geranylgeranyled on either one or two cysteine residues in their C-terminus to localize to the correct intracellular membrane.

[0053] In some embodiments, a prenylation reaction can be reproduced in vitro to test for REP1 potency. In some embodiments, a substrate for a prenylation assay is a Rab GTPase. In some embodiments, a substrate for a prenylation assay is a Rab GTPase portion or fragment thereof. In some embodiments, a substrate for a prenylation assay is a protein comprising a Rab GTPase portion or fragment thereof .In some embodiments, a substrate for a prenylation assay is Rab27a. The Rab27a protein was first identified in the cytosolic fraction of CHM lymphoblasts in 1995. In some embodiments, a substrate for a prenylation assay is RAB6A. In some embodiments, the response of, Rab27A and/or RAB6A, to the incorporation of a biotinylated lipid donor in a prenylation reaction can be assayed in vitro and used to develop robust and sensitive assays for assessing the biological activity of AAV vectors for choroideremia.

[0054] An example amino acid sequence of REP1 is:

[0055] MADTLPSEFDVIVIGTGLPESIIAAACSRSGRRVLHVDSRSYYGGNWA

SFSFSGLLSWLKEYQENSDIVSDSPVWQDQILENEEAIALSRKDTIQHVEVFCYASQ D EHEDVEEAGAEQKNHAEVTSANSTEAADSAFLPTEDESLSTMSCEMLTEQTPSSDPE NALEVNGAEVTGEKENHCDDKTCVPSTSAEDMSENVPIAEDTTEQPKKNRITYSQII EGRRFNIDEVSKEEYSRGEEIDLLIKSNVSRYAEF NITRIEAFREGRVEQVPCSRADVFNSKQETMVEKRMLMKFLTFCMEYEKYPDEY GYEEITFYEYEKTQKETPNEQYIVMHSIAMTSETASSTIDGLKATKNFLHCLGRYGN TPFEFPEYGQGELPQCFCRMCAVFGGIYCLRHSVQCLVVDKESRKCKAIIDQFGQRII SEHFEVEDSYFPENMCSRVQYRQISRAVEITDRSVLTDSDQQISILTVPAEEPGTFAV RVIEECSSTMTCMKGTYEVHLTCTSSKTAREDLESVVQKLFVPYTEMEIENEQVEKP RIEWAEYFNMRDSSDISRSCYNDLPSNVYVCSGPDCGLGNDNAVKQAETLFQEICP NEDFCPPPPNPEDIIEDGDSLQPEASESSAIPEANSETFKESTNLGNLEESSE (SEQ ID NO: 1).

[0056] An example amino acid sequence of REP1 is:

MADTEPSEFDVIVIGTGEPESIIAAACSRSGRRVEHVDSRSYYGGNWASFSFSGLLS W EKEYQENSDIVSDSPVWQDQIEENEEAIALSRKDKTIQHVEVFCYASQDLHEDVEEA GAEQKNHAEVTSANSTEAADSAFEPTEDESLSTMSCEMLTEQTPSSDPENALEVNG AEVTGEKENHCDDKTCVPSTSAEDMSENVPIAEDTTEQPKKNRITYSQIIKEGRRFNI DEVSKEEYSRGEEIDEEIKSNVSRYAEFKNITRILAFREGRVEQVPCSRADVFNSKQL TMVEKRMEMKFETFCMEYEKYPDEYKGYEEITFYEYLKTQKLTPNLQYIVMHSIA MTSETASSTIDGEKATKNFEHCEGRYGNTPFEFPLYGQGELPQCFCRMCAVFGGIYC ERHSVQCEVVDKESRKCKAIIDQFGQRIISEHFLVEDSYFPENMCSRVQYRQISRAVL ITDRSVEKTDSDQQISIETVPAEEPGTFAVRVIELCSSTMTCMKGTYLVHLTCTSSKT AREDEESVVQKEFVPYTEMEIENEQVEKPRIEWALLFNMRDSSDISRSCYNDEPSNV YVCSGPDCGLGNDNAVKQAETLFQEICPNEDFCPPPPNPEDIILDGDSLQPEASESSAI

PEANSETFKESTNLGNLEESSE (SEQ ID NO: 2).

[0057] An example nucleotide sequence encoding REP1 is:

[0058] ATGGCGGATACTCTCCCTTCGGAGTTTGATGTGATCGTAATAGGG

ACGGGTTTGCCTGAATCCATCATTGCAGCTGCATGTTCAAGAAGTGGCCGGAGA

GTTCTGCATGTTGATTCAAGAAGCTACTATGGAGGAAACTGGGCCAGTTTTAGC

TTTTCAGGACTATTGTCCTGGCTAAAGGAATACCAGGAAAACAGTGACATTGTA

AGTGACAGTCCAGTGTGGCAAGCCGATCCTTGAAAATGAAG

AGCCATTGCTCTTAGCAGGAAGGACAAAACATTCAACATGTGGAAGTATTTTGT

TATGCCAGTCAGGATTTGCATGAAGATGTCGAAGAAGCTGGTGCACTGCAGAAA

AATCATGCTCTTGTGACATCTGCAAACTCCACAGAAGCTGCAGATTCTGCCTTCC

TGCCTACGGAGGATGAGTCATTAAGCACTATGAGCTGTGAAATGCTCACAGAAC

AAACTCCAAGCAGCGATCCAGAGAATGCGCTAGAAGTAAATGGTGCTGAAGTG

ACAGGGGAAAAAGAAAACCATTGTGATGATAAAACTTGTGTGCCATCAACTTCA

GCAGAAGACATGAGTGAAAATGTGCCTATAGCAGAAGATACCACAGAGCAACC

AAAGAAAAACAGAATTACTTACTCACAAATTATTAAAGAAGGCAGGAGATTTA

ATATTGATTTAGTATCAAAGCTGCTGTATTCTCGAGGATTACTAATTGATCTTCT

AATCAAATCTAATGTTAGTCGATATGCAGAGTTTAAAAATATTACCAGGATTCT

TGCATTTCGAGAAGGCGAGTGGAACAGGTTCCGTGTTCCGGCGATGTCTTTAAT

AGCAAACAACTTACTATGGTAGAAAAGCGAATGCTAATGAAATTTCTTACATTT

TGTATGGAATATGAGAAATATCCTGATGAATAT

AAAGGATATGAAGAGATCACATTTTTGAATTTTAAAGACTCAAAAATTAACCCC

CAACCTCCAATATATTGTCATGCATTCAATTGCAATGACATCAGAGACAGCCAG

CAGCACCATAGATGGTCTCAAAGCTACCAAAAACTTTCTTCACTGTCTTGGGCG

GTATGGCAACACTCCATTTTTGTTTCCTTTATATGGCCAAGGAGAACTCCCCCAG

TGTTTCTGCAGGATGTGTGCTGTGTTTGGTGGAATTTATTGTCTTCGCCATTCAG

TACAGTGCCTTGTAGTGGACAAAGAATCCAGAAAATGTAAAGCAATTATAGATC

AGTTTGGTCAGAGAATAATCTCTGAGCATTTCCTCGTGGAGGACAGTTACTTTCC

TGAGAACATGTGCTCACGTGTGCAATACAGGCAGATCTCCAGGGCAGTGCTGAT

TACAGAAGATCTGTCCTAAAAACAGATTCAGATCAACGATTTCCTTTTGACAGT GCCAGCAGAGGAACCAGGAACTTTTGCTGTTCGGGTCATTGAGTTATGTTCTTC

AACGATGACATGCATGAAAGGCACCTATTTGGTTCATTTGACTTGCACATCTTCT

AAAACAGCAAGAGAAGATTTAGAATCAGTTGTGCAGAAATTGTTTGTTCCATAT

ACTGAAATGGAGATAGAAAATGAACAAGTAGAAAAGCCAAGAATTCTGTGGGC

TCTTTACTTCAATATGAGAGATTCGTCAGACATCAGCAGGAGCTGTTATAATGA

TTTACCATCCAACGTTTATGTCTGCTCTGGCCCAGATTGTGGTTTAGGAAATGAT

AATGCAGTCAAACAGGCTGAAACACTTTTCCAGGAAATCTGCCCCAATGAAGAT

TTCTGTCCCCCTCCCCAAATCCTGAAGACATTATCCTTGATGGAGACAGTTTACA GCCAGAG

GCTTCAGAATCCAGTGCCATACCAGAGGCTAACTCGGAGACTTTCAAGGAAAGC ACAAACCTTGGAAACCTAGAGGAGTCCTCTGAAAA (SEQ ID NO: 3)

[0059] A further example nucleotide sequence encoding REP1 is:

[0060] GATATCGAATTCCTGCAGCCCGGCGGCACCATGGCGGATACTCTC

CCTTCGGAGTTTGATGTGATCGTAATAGGGACGGGTTTGCCTGAATCCATCATTG

CAGCTGCATGTTCAAGAAGTGGCCGGAGAGTTCTGCATGTTGATTCAAGAAGCT

ACTATGGAGGAAACTGGGCCAGTTTTAGCTTTTCAGGACTATTGTCCTGGCTAA

AGGAATACCAGGAAAACAGTGACATTGTAAGTGACAGTCCAGTGTGGCAAGAC

CAGATCCTTGAAAATGAAGAAGCCATTGCTCTTAGCAGGAAGGACAAAACTATT

CAACATGTGGAAGTATTTTGTTATGCCAGTCAGGATTTGCATGAAGATGTCGAA

GAAGCTGGTGCACTGCAGAAAAATCATGCTCTTGTGACATCTGCAAACTCCACA

GAAGCTGCAGATTCTGCCTTCCTGCCTACGGAGGATGAGTCATTAAGCACTATG

AGCTGTGAAATGCTCACAGAACAAACTCCAAGCAGCGATCCAGAGAATGCGCT

AGAAGTAAATGGTGCTGAAGTGACAGGGGAAAAAGAAAACCATTGTGATGATA

AAACTTGTGTGCCATCAACTTCAGCAGAAGACATGAGTGAAAATGTGCCTATAG

CAGAAGATACCACAGAGCAACCAAAGAAAAACGAATTACTTACTCACAAATAT

TAAGAAGGCAGGAGATTAATATTGATTTAGTATCAAAGCTGCTGTATTCTCGAG

GATTACTAATTGATCTTCTAATCAAATCTAATGTTAGTCGATATGCAGAGTTTAA

AAATATTACCAGGATTCTTGCATTTCGAGAAGGACGAGTGGAACAGGTTCCGTG

TTCCAGAGCAGATGTCTTTAATAGCAAACAACTTACTATGGTAGAAAAGCGAAT

GCTAATGAAATTTCTTACATTTTGTATGGAATATGAGAAATATCCTGATGAATAT AAAGGATATGAAGAGATCACATTTTATGAATATTTAAAGACTCAAAAATTAACC

CCCAACCTCCAATATATTGTCATGCATTCAATTGCAATGACATCAGAGACAGCC

AGCAGCACCATAGATGGTCTCAAAGCTACCAAAAACTTTCTTCACTGTCTTGGG

CGGTATGGCAACACTCCATTTTTGTTTCCTTTATATGGCCAAGGAGAACTCCCCC

AGTGTTTCTGCAGGATGTGTGCTGTGTTTGGTGGAATTTATTGTCTTCGCCATTC

AGTACAGTGCCTTGTAGTGGACAAAGAATCCAGAAAATGTAAAGCAATTATAG

ATCAGTTTGGTCAGAGAATAATCTCTGAGCATTTCCTCGTGGAGGACAGTTACTT

TCCTGAGAACATGTGCTCACGT

GTGCAATACAGGCAGATCTCCAGGGCAGTGCTGATTACAGATAGATCTGTCCTA

AAAACAGATTCAGATCAACAGATTTCCATTTTGACAGTGCCAGCAGAGGAACCA

GGAACTTTTGCTGTTCGGGTCATTGAGTTATGTTCTTCAACGATGACATGCTGAA

AGGCACCTATTTGGTTCATTTGACTTGCACATCTTCTAAAACAGCAAGAGAAGA

TTTAGAATCAGTTGTGCAGAAATTGTTTGTTCCATATACTGAAATGGAGATAGA

AAATGAACAAGTAGAAAAGCCAAGAATTCTGTGGGCTCTTTACTTCAATATGAG

AGATTCGTCAGACATCAGCAGGAGCTGTTATAATGATTTACCATCCAACGTTTA

TGTCTGCTCTGGCCCAGATTGTGGTTTAGGAAATGATAATGCAGTCAAACAGGC

TGAAACACTTTTCCAGGAAATCTGCCCCAATGAAGATTTCTGTCCCCCTCCACCA

AATCCTGAAGACATTATCCTTGATGGAGACAGTTTACAGCCAGAGGCTTCAGAA

TCCAGTGCCATACCAGAGGCTAACTCGGAGACTTTCAGGAAAGCACAAACCTTG

GAAACCTAGAGGAGTCCTCTGAA AA (SEQ ID NO: 4)

[0061] A further example nucleotide sequence encoding REP1 is:

ATGGCGGATACTCTCCCTTCGGAGTTTGATGTGATCGTAATAGGGACGGGTTTG

CCTGAATCCATCATTGCAGCTGCATGTTCAAGAAGTGGCCGGAGAGTTCTGCAT

GTTGATTCAAGAAGCTACTATGGAGGAAACTGGGCCAGTTTTAGCTTTTCAGGA

CTATTGTCCTGGCTAAAGGAATACCAGGAAAACAGTGACATTGTAAGTGACAGT

CCAGTGTGGCAAGACCAGATCCTTGAAAATGAAGAAGCCATTGCTCTTAGCAGG

AAGGACAAAACTATTCAACATGTGGAAGTATTTTGTTATGCCAGTCAGGATTTG

CATGAAGATGTCGAAGAAGCTGGTGCACTGCAGAAAAATCATGCTCTTGTGACA

TCTGCAAACTCCACAGAAGCTGCAGATTCTGCCTTCCTGCCTACGGAGGATGAG

TCATTAAGCACTATGAGCTGTGAAATGCTCACAGAACAAACTCCAAGCAGCGAT

CCAGAGAATGCGCTAGAAGTAAATGGTGCTGAAGTGACAGGGGAAAAAGAAAA CCATTGTGATGATAAAACTTGTGTGCCATCAACTTCAGCAGAAGACATGAGTGA

AAATGTGCCTATAGCAGAAGATACCACAGAGCAACCAAAGAAAAACAGAATTA

CTTACTCACAAATTATTAAAGAAGGCAGGAGATTTAATATTGATTTAGTATCAA

AGCTGCTGTATTCTCGAGGATTACTAATTGATCTTCTAATCAAATCTAATGTTAG

TCGATATGCAGAGTTTAAAAATATTACCAGGATTCTTGCATTTCGAGAAGGACG

AGTGGAACAGGTTCCGTGTTCCAGAGCAGATGTCTTTAATAGCAAACAACTTAC

TATGGTAGAAAAGCGAATGCTAATGAAATTTCTTACATTTTGTATGGAATATGA

GAAATATCCTGATGAATATAAAGGATATGAAGAGATCACATTTTATGAATATTT

AAAGACTCAAAAATTAACCCCCAACCTCCAATATATTGTCATGCATTCAATTGC

AATGACATCAGAGACAGCCAGCAGCACCATAGATGGTCTCAAAGCTACCAAAA

ACTTTCTTCACTGTCTTGGGCGGTATGGCAACACTCCATTTTTGTTTCCTTTATAT

GGCCAAGGAGAACTCCCCCAGTGTTTCTGCAGGATGTGTGCTGTGTTTGGTGGA

ATTTATTGTCTTCGCCATTCAGTACAGTGCCTTGTAGTGGACAAAGAATCCAGA

AAATGTAAAGCAATTATAGATCAGTTTGGTCAGAGAATAATCTCTGAGCATTTC

CTCGTGGAGGACAGTTACTTTCCTGAGAACATGTGCTCACGTGTGCAATACAGG

CAGATCTCCAGGGCAGTGCTGATTACAGATAGATCTGTCCTAAAAACAGATTCA

GATCAACAGATTTCCATTTTGACAGTGCCAGCAGAGGAACCAGGAACTTTTGCT

GTTCGGGTCATTGAGTTATGTTCTTCAACGATGACATGCATGAAAGGCACCTATT

TGGTTCATTTGACTTGCACATCTTCTAAAACAGCAAGAGAAGATTTAGAATCAG

TTGTGCAGAAATTGTTTGTTCCATATACTGAAATGGAGATAGAAAATGAACAAG

TAGAAAAGCCAAGAATTCTGTGGGCTCTTTACTTCAATATGAGAGATTCGTCAG

ACATCAGCAGGAGCTGTTATAATGATTTACCATCCAACGTTTATGTCTGCTCTGG

CCCAGATTGTGGTTTAGGAAATGATAATGCAGTCAAACAGGCTGAAACACTTTT

CCAGGAAATCTGCCCCAATGAAGATTTCTGTCCCCCTCCACCAAATCCTGAAGA

CATTATCCTTGATGGAGACAGTTTACAGCCAGAGGCTTCAGAATCCAGTGCCAT

ACCAGAGGCTAACTCGGAGACTTTCAAGGAAAGCACAAACCTTGGAAACCTAG AGGAGTCCTCTGAATAA (SEQ ID NO: 5).

[0062] A further example nucleotide sequence encoding REP1 is:

GATATCGAATTCCTGCAGCCCGGCGGCACCATGGCGGATACTCTCCCTTCGGAG

TTTGATGTGATCGTAATAGGGACGGGTTTGCCTGAATCCATCATTGCAGCTGCAT

GTTCAAGAAGTGGCCGGAGAGTTCTGCATGTTGATTCAAGAAGCTACTATGGAG GAAACTGGGCCAGTTTTAGCTTTTCAGGACTATTGTCCTGGCTAAAGGAATACC

AGGAAAACAGTGACATTGTAAGTGACAGTCCAGTGTGGCAAGACCAGATCCTT

GAAAATGAAGAAGCCATTGCTCTTAGCAGGAAGGACAAAACTATTCAACATGT

GGAAGTATTTTGTTATGCCAGTCAGGATTTGCATGAAGATGTCGAAGAAGCTGG

TGCACTGCAGAAAAATCATGCTCTTGTGACATCTGCAAACTCCACAGAAGCTGC

AGATTCTGCCTTCCTGCCTACGGAGGATGAGTCATTAAGCACTATGAGCTGTGA

AATGCTCACAGAACAAACTCCAAGCAGCGATCCAGAGAATGCGCTAGAAGTAA

ATGGTGCTGAAGTGACAGGGGAAAAAGAAAACCATTGTGATGATAAAACTTGT

GTGCCATCAACTTCAGCAGAAGACATGAGTGAAAATGTGCCTATAGCAGAAGA

TACCACAGAGCAACCAAAGAAAAACAGAATTACTTACTCACAAATTATTAAAG

AAGGCAGGAGATTTAATATTGATTTAGTATCAAAGCTGCTGTATTCTCGAGGAT

TACTAATTGATCTTCTAATCAAATCTAATGTTAGTCGATATGCAGAGTTTAAAAA

TATTACCAGGATTCTTGCATTTCGAGAAGGACGAGTGGAACAGGTTCCGTGTTC

CAGAGCAGATGTCTTTAATAGCAAACAACTTACTATGGTAGAAAAGCGAATGCT

AATGAAATTTCTTACATTTTGTATGGAATATGAGAAATATCCTGATGAATATAA

AGGATATGAAGAGATCACATTTTATGAATATTTAAAGACTCAAAAATTAACCCC

CAACCTCCAATATATTGTCATGCATTCAATTGCAATGACATCAGAGACAGCCAG

CAGCACCATAGATGGTCTCAAAGCTACCAAAAACTTTCTTCACTGTCTTGGGCG

GTATGGCAACACTCCATTTTTGTTTCCTTTATATGGCCAAGGAGAACTCCCCCAG

TGTTTCTGCAGGATGTGTGCTGTGTTTGGTGGAATTTATTGTCTTCGCCATTCAG

TACAGTGCCTTGTAGTGGACAAAGAATCCAGAAAATGTAAAGCAATTATAGATC

AGTTTGGTCAGAGAATAATCTCTGAGCATTTCCTCGTGGAGGACAGTTACTTTCC

TGAGAACATGTGCTCACGTGTGCAATACAGGCAGATCTCCAGGGCAGTGCTGAT

TACAGATAGATCTGTCCTAAAAACAGATTCAGATCAACAGATTTCCATTTTGAC

AGTGCCAGCAGAGGAACCAGGAACTTTTGCTGTTCGGGTCATTGAGTTATGTTC

TTCAACGATGACATGCATGAAAGGCACCTATTTGGTTCATTTGACTTGCACATCT

TCTAAAACAGCAAGAGAAGATTTAGAATCAGTTGTGCAGAAATTGTTTGTTCCA

TATACTGAAATGGAGATAGAAAATGAACAAGTAGAAAAGCCAAGAATTCTGTG

GGCTCTTTACTTCAATATGAGAGATTCGTCAGACATCAGCAGGAGCTGTTATAA

TGATTTACCATCCAACGTTTATGTCTGCTCTGGCCCAGATTGTGGTTTAGGAAAT

GATAATGCAGTCAAACAGGCTGAAACACTTTTCCAGGAAATCTGCCCCAATGAA

GATTTCTGTCCCCCTCCACCAAATCCTGAAGACATTATCCTTGATGGAGACAGTT TACAGCCAGAGGCTTCAGAATCCAGTGCCATACCAGAGGCTAACTCGGAGACTT

TCAAGGAAAGCACAAACCTTGGAAACCTAGAGGAGTCCTCTGAATAA (SEQ ID NO: 6).

[0063] Example variants of REP1 are described further in WO 2012/114090 (incorporated herein by reference).

Viral Vectors

[0064] Choroideremia may be treated by providing functional copies of the CHM gene (REP1) to the affected cells of the eye. In some embodiments, REP1 can be delivered to affected cells of the eye using a recombinant viral vector. In some embodiments, a recombinant viral vector is a lentiviral vector; an adenoviral vector, or an adeno-associated viral vector. In some embodiments a recombinant viral vector is an adeno-associated viral vector. In some embodiments, a viral vector is, a recombinant adeno-associated virus (rAAV) vector encoding REP1. In some embodiments a recombinant viral vector is a serotype two adeno-associated viral vector (rAAV2). In some embodiments, a recombinant viral vector encodes a payload. In some embodiments, a payload is a protein. In some embodiments, a recombinant viral vector is, a serotype 2 adeno-associated virus (rAAV2) vector encoding human REP1 (rAAV2-hREP1).

[0065] In some embodiments a viral vector is added to cell culture at one or more (e.g., multiple) multiplicities of infection (MOI). In some embodiments, transduction with a viral vector is performed at more than one different MOI, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more MOIs, e.g., 2-10, 3-10, or 4-10 different MOIs. In some embodiments, one or more MOIs are selected that produce a signal to noise ratio in the assay results of at least 2, e.g., at least 2.5, 3, 4, 5, 6, 7, 8, 9, 10 or more. In some embodiments, one or more MOIs are selected that produce a linear range of assay results (e.g., level of expression of protein or enzymatic activity of protein). In some embodiments, assaying multiple MOIs is achieved by serial dilution. In some embodiments serial dilutions are achieved by about10 fold, 9 fold, 8 fold, 7 fold, 6 fold, 5 fold, 4 fold, 3 fold, 2.8 fold, 2.5 fold, 2 fold, 1.8 fold, or 1.5 fold, or 0.5 fold to 3 fold, or 0.5 to 6 fold, or 0.5 to 8 fold, or 0.5 to 10 fold, serial dilutions. [0066] Viral titer can be measured by various methods. In some embodiments the titer of a viral vector is determined by qPCR. In some embodiments the titer of a viral vector is determined by droplet digital PCR (ddPCR e.g., as described in Lock et al., Hum Gene Ther Methods. 2014 Apr;25(2): 115-25).

[0067] In some embodiments, a viral vector titer (determined by, e.g., qPCR or ddPCR) is expressed as viral genomes (VG) per a volume (e.g., VG/ml, VG/μL) or viral genomes per cell (VG/cell). In some embodiments, a titer describing viral genomes (VG) is equivalent to a titer expressed as DNase Resistant Particles (DRP). In some embodiments, a titer describing viral genomes (VG) is equivalent to a titer expressed as genome particles (GP).

[0068] In some embodiments, a viral vector is added to cell culture at an MOI of about 500,000 vg/cell to about 20 vg/cell. In some embodiments, a viral vector is added to cell culture at an MOI of about 400,000 vg/cell to about 1,000 vg/cell. In some embodiments, a viral vector is added to cell culture at an MOI of about 300,000 vg/cell to about 1,000 vg/cell. In some embodiments, a viral vector is added to cell culture at an MOI of about 200,000 vg/cell to about 1,000 vg/cell. In some embodiments, a viral vector is added to cell culture at an MOI of about 100,000 vg/cell to about 1,000 vg/cell. In some embodiments, a viral vector is added to cell culture at an MOI of about 200,000 vg/cell to about 90,000 vg/cell. In some embodiments, a viral vector is added to cell culture at an MOI of about 100,000 vg/cell to about 80,000 vg/cell. In some embodiments, a viral vector is added to cell culture at an MOI of about 90,000 vg/cell to about 45,000 vg/cell. In some embodiments, a viral vector is added to cell culture at an MOI of about 90,000 vg/cell to about 70,000 vg/cell. In some embodiments, a viral vector is added to cell culture at an MOI of about 80,000 vg/cell to about 60,000 vg/cell. In some embodiments, a viral vector is added to cell culture at an MOI of about 50,000 vg/cell to about 70,000 vg/cell. In some embodiments, a viral vector is added to cell culture at an MOI of about 50,000 vg/cell to about 25,000 vg/cell. In some embodiments, a viral vector is added to cell culture at an MOI of about 40,000 vg/cell to about 60,000 vg/cell. In some embodiments, a viral vector is added to cell culture at an MOI of about 30,000 vg/cell to about 10,000 vg/cell. In some embodiments, a viral vector is added to cell culture at an MOI of about 30,000 vg/cell to about 50,000 vg/cell. In some embodiments, a viral vector is added to cell culture at an MOI of about 20,000 vg/cell to about 8,000 vg/cell. In some embodiments, a viral vector is added to cell culture at an MOI of about 40,000 vg/cell to about 20,000 vg/cell. In some embodiments, a viral vector is added to cell culture at an MOI of about 30,000 vg/cell to about 10,000 vg/cell. In some embodiments, a viral vector is added to cell culture at an MOI of about 15,000 vg/cell to about 9,000 vg/cell. In some embodiments, a viral vector is added to cell culture at an MOI of about 8,000 vg/cell to about 1,500 vg/cell. In some embodiments, a viral vector is added to cell culture at an MOI of about 2,000 vg/cell to about 500 vg/cell.

[0069] In some embodiments, a viral vector is added to cell culture at an MOI of about 366,666 vg/cell +/- 50%; 366,666 vg/cell +/- 40%; 366,666 vg/cell +/- 30%; 366,666 vg/cell +/- 20%; 366,666 vg/cell +/- 10%; 366,666 vg/cell +/- 5%; or 366,666 vg/cell +/- 1%, e.g., where the titer of the viral vector has been determined by qPCR; or an MOI equivalent to these aforementioned MOIs where the titer has been determined by another method.

[0070] In some embodiments, a viral vector is added to cell culture at an MOI of about 183,000 vg/cell +/- 50%; 183,000 vg/cell +/- 40%; 183,000 vg/cell +/- 30%; 183,000 vg/cell +/- 20%; 183,000 vg/cell +/- 10%; 183,000 vg/cell +/- 5%; or 183,000 vg/cell +/- 1%, e.g., where the titer of the viral vector has been determined by qPCR; or an MOI equivalent to these aforementioned MOIs where the titer has been determined by another method.

[0071] In some embodiments, a viral vector is added to cell culture at an MOI of about 73,200 vg/cell +/- 50%; 73,200 vg/cell +/- 40%; 73,200 vg/cell +/- 30%; 73,200 vg/cell +/- 20%; 73,200 vg/cell +/- 10%; 73,200 vg/cell +/- 5%; or 73,200 vg/cell +/- 1%, e.g., where the titer of the viral vector has been determined by qPCR; or an MOI equivalent to these aforementioned MOIs where the titer has been determined by another method.

[0072] In some embodiments, a viral vector is added to cell culture at an MOI of about 29,300 vg/cell +/- 50%; 29,300 vg/cell +/- 40%; 29,300 vg/cell +/- 30%; 29,300 vg/cell +/- 20%; 29,300 vg/cell +/- 10%; 29,300 vg/cell +/- 5%; or 29,300 vg/cell +/- 1%, e.g., where the titer of the viral vector has been determined by qPCR; or an MOI equivalent to these aforementioned MOIs where the titer has been determined by another method. [0073] In some embodiments, a viral vector is added to cell culture at an MOI of about 11,700 vg/cell +/- 50%; 11,700 vg/cell +/- 40%; 11,700 vg/cell +/- 30%; 11,700 vg/cell +/- 20%; 11,700 vg/cell +/- 10%; 11,700 vg/cell +/- 5%; or 11,700 vg/cell +/- 1%, e.g., where the titer of the viral vector has been determined by qPCR; or an MOI equivalent to these aforementioned MOIs where the titer has been determined by another method. [0074] In some embodiments, a viral vector is added to cell culture at an MOI of about 4,680 vg/cell +/- 50%; 4,680 vg/cell +/- 40%; 4,680 vg/cell +/- 30%; 4,680 vg/cell +/- 20%; 4,680 vg/cell +/- 10%; 4,680 vg/cell +/- 5%; or 4,680 vg/cell +/- 1%, e.g., where the titer of the viral vector has been determined by qPCR; or an MOI equivalent to these aforementioned MOIs where the titer has been determined by another method.

[0075] In some embodiments, a viral vector is added to cell culture at an MOI of about 780vg/cell +/- 50%; 780vg/cell +/- 40%; 780vg/cell +/- 30%; 780vg/cell +/- 20%; 780vg/cell +/- 10%; 780vg/cell +/- 5%; or 780vg/cell +/- 1%, e.g., where the titer of the viral vector has been determined by qPCR; or an MOI equivalent to these aforementioned MOIs where the titer has been determined by another method.

[0076] In some embodiments, a viral vector is added to cell culture at an MOI of about 130vg/cell +/- 50%; 130vg/cell +/- 40%; 130vg/cell +/- 30%; 130vg/cell +/- 20%; 130vg/cell +/- 10%; 130vg/cell +/- 5%; or 130vg/cell +/- 1%, e.g., where the titer of the viral vector has been determined by qPCR; or an MOI equivalent to these aforementioned MOIs where the titer has been determined by another method.

[0077] In some embodiments, a viral vector is added to cell culture at an MOI of about 140,000vg/cell +/- 50%; 140,000vg/cell +/- 40%; 140,000vg/cell +/- 30%; 140,000vg/cell +/- 20%; 140,000vg/cell +/- 10%; 140,000vg/cell +/- 5%; or 140,000vg/cell +/- 1%, e.g., where the titer of the viral vector has been determined by qPCR; or an MOI equivalent to these aforementioned MOIs where the titer has been determined by another method.

[0078] In some embodiments, a viral vector is added to cell culture at an MOI of about 76,000vg/cell +/- 50%; 76,000vg/cell +/- 40%; 76,000vg/cell +/- 30%; 76,000vg/cell +/- 20%; 76,000vg/cell +/- 10%; 76,000vg/cell +/- 5%; or 76,000vg/cell +/- 1%, e.g., where the titer of the viral vector has been determined by qPCR; or an MOI equivalent to these aforementioned MOIs where the titer has been determined by another method. [0079] In some embodiments, a viral vector is added to cell culture at an MOI of about 42,000vg/cell +/- 50%; 42,000vg/cell +/- 40%; 42,000vg/cell +/- 30%; 42,000vg/cell +/- 20%; 42,000vg/cell +/- 10%; 42,000vg/cell +/- 5%; or 42,000vg/cell +/- 1%, e.g., where the titer of the viral vector has been determined by qPCR; or an MOI equivalent to these aforementioned MOIs where the titer has been determined by another method.

[0080] In some embodiments, a viral vector is added to cell culture at an MOI of about 24,000vg/cell +/- 50%; 24,000vg/cell +/- 40%; 24,000vg/cell +/- 30%; 24,000vg/cell +/- 20%; 24,000vg/cell +/- 10%; 24,000vg/cell +/- 5%; or 24,000vg/cell +/- 1%, e.g., where the titer of the viral vector has been determined by qPCR; or an MOI equivalent to these aforementioned MOIs where the titer has been determined by another method.

[0081] In some embodiments, a viral vector is added to cell culture at an MOI of about 13,000vg/cell +/- 50%; 13,000vg/cell +/- 40%; 13,000vg/cell +/- 30%; 13,000vg/cell +/- 20%; 13,000vg/cell +/- 10%; 13,000vg/cell +/- 5%; or 13,000vg/cell +/- 1%, e.g., where the titer of the viral vector has been determined by qPCR; or an MOI equivalent to these aforementioned MOIs where the titer has been determined by another method.

[0082] In some embodiments, MOIs are selected that produce a linear range of biological activity assay results. In other embodiments, MOIs are selected that produce a non-linear range of biological activity assay results. In some embodiments, a signal to noise ratio of a biological activity assay is at least about 2, e.g., at least about 2.5 or greater than

2.5, e.g., a signal to noise ratio of about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about

5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, about 10, or greater. In some embodiments, a signal to noise ratio of about 2.5 or greater allows for use of a non-linear range of biological activity assay results.

[0083] In some embodiments, at least 4, 5, 6, 7, 8, 9, or 10 different MOIs (e.g., 4, 5, 6, 7, 8, 9, or 10 different MOIs) are tested, e.g., by serial dilution. In some embodiments serial dilutions are achieved by about 10 fold, 9 fold, 8 fold, 7 fold, 6 fold, 5 fold, 4 fold, 3 fold, 2.8 fold, 2.5 fold, 2 fold, 1.8 fold, or 1.5 fold, or 0.5 fold to 3 fold, or 0.5 to 6 fold, or 0.5 to 8 fold, or 0.5 to 10 fold, serial dilutions. In some embodiments, an initial (e.g., high) MOI, prior to serial dilution is an MOI described herein. In some embodiments, e.g., for an enzymatic activity assay described herein, an initial MOI is an MOI described herein (e.g., about 140,000 vg/cell, where the titer has been determined by qPCR; or an equivalent MOI where the titer has been determined by another method), and multiple serial dilutions are tested at about 1.8 fold for each dilution, e.g., down to a MOI of about 13,000 vg/cell (e.g., where the titer has been determined by qPCR; or an equivalent MOI where the titer has been determined by another method). In some embodiments, e.g., for an enzymatic activity assay described herein, an initial MOI is about 94,000 vg/cell (e.g., where the titer has been determined by ddPCR; or an equivalent MOI where the titer has been determined by another method), and multiple serial dilutions are tested at about 1.8 fold for each dilution, e.g., down to a MOI of about 9,000 vg/cell (e.g., where the titer has been determined by ddPCR; or an equivalent MOI where the titer has been determined by another method). In some embodiments, e.g., for an expression assay described herein, an initial MOI is about 366,000 vg/cell (e.g., where the titer has been determined by qPCR; or an equivalent MOI where the titer has been determined by another method), and multiple serial dilutions are tested, e.g., down to a MOI of about 130 vg/cell (e.g., where the titer has been determined by qPCR; or an equivalent MOI where the titer has been determined by another method). In some embodiments, e.g., for an expression assay described herein, an initial MOI is about 79,800 vg/cell (e.g., where the titer has been determined by ddPCR; or an equivalent MOI where the titer has been determined by another method), and multiple serial dilutions are tested, e.g., down to a MOI of about 28.4 vg/cell (e.g., where the titer has been determined by ddPCR; or an equivalent MOI where the titer has been determined by another method).

[0084] In some embodiments an MOI used depends on the method used to determine the viral titer (e.g., qPCR or ddPCR or another suitable method). In some embodiments, when determining the activity (e.g., enzymatic) of a protein encoded by the recombinant viral vector and the titer of the viral vector is determined by ddPCR, a viral vector is added to cell culture at an MOI(s) from about 9.4E4 vg/cell to about 9E3 vg/cell or about 5E3 vg/cell. In some embodiments, when determining the activity (e.g., enzymatic) of a protein encoded by the recombinant viral vector and the titer of the viral vector is determined by qPCR, a viral vector is added to cell culture at an MOI(s) from about 5E5 vg/cell or about 1.4E5 vg/cell to about 1.3E4 vg/cell. In some embodiments, when determining the level of expression of a payload (e.g., a protein) encoded by the recombinant viral vector and the titer of the viral vector is determined by ddPCR, a viral vector is added to cell culture at an MOI(s) from about 7.98E4 vg/cell to about 2.84E1 vg/cell. In some embodiments, when determining the level of expression of a payload (e.g., a protein) encoded by the recombinant viral vector and the titer of the viral vector is determined by qPCR, a viral vector is added to cell culture at an MOI(s) from about 3.66E5 vg/cell to about 1.3E2 vg/cell.

[0085] Equivalent MOIs to those described herein can be used if methods other than qPCR or ddPCR have been used to determine the titer. One of skill in the art should be able to convert the equivalent MOI values accordingly based on how titer has been determined.

[0086] MOI values described herein can be used for a potency assay described herein, e.g., an enzymatic activity based potency assay described herein or an expression- based potency assay described herein.

Cells

[0087] The present disclosure recognizes that cells vary in permissivity to viral vectors. Further, the present disclosure recognizes that cells vary in their capability to transcribe and translate proteins encoded by viral vectors. Thus, without wishing to be bound by any particular theory, the present disclosure recognizes that cells used for biological activity assays as described herein must be highly permissive to viral vectors. Thus, in accordance with various embodiments, assays as described herein utilize a cell permissive for a viral vector encoding a protein of interest.

[0088] In some embodiments, a cell is mammalian cell. In some embodiments, a cell is a human cell. In some embodiments, a cell is an immortalized cell. In some embodiments, a cell comprises a SV40 large T antigen. In some embodiments, a cell is derived from kidney tissue. In some embodiments, a cell is derived from liver tissue. In some embodiments a cell is derived from eye tissue. In some embodiments a cell is a HuH7 cell. In some embodiments, a cell is a HEK293 cell. In some embodiments, a cell is a HEK293T cell. In some embodiments a cell is a retinal pigmented epithelial cell (e.g., RPE-1). In some embodiments a cell is not a retinal pigmented epithelial cell.

[0089] In some embodiments, cells are seeded and transduced on the same day. In some embodiments, cells are thawed, seeded, and transduced on the same day. In some embodiments, cells are cultured for 3, 4, 5, 6, 7, or 8 hours prior to transduction with a viral vector. In some embodiments, cells are seeded onto a substrate (e.g., a cell culture vessel) prior to transduction with a recombinant viral vector described herein. Cells can be cultured in a cell culture vessel. Cell culture vessels can comprise a cell culture dish, plate, or flask. Exemplary cell culture vessels include 35mm, 60mm, 100mm, or 150mm dishes, multi-well plates (e.g., 6-well, 12-well, 24-well, 48-well, or 96 well plates), or flasks (e.g., T-flasks, e.g., T-25, T-75, or T-160 flasks), or shaker flasks. In some embodiments cells are seeded in 96 well plates. In some embodiments, cells are plated to achieve a confluency of about 30% to about 70% (e.g., about 30-40%, 40-50%, 50-60%, 60-70%, 30-50%, 40-70%, about 30%, about 40%, about 50%, about 60%, or about 70%) at the time of transduction with a recombinant viral vector.

[0090] In some embodiments, cells are plated, e.g., in a 96-well plate, at a density of about 1E+4 to about 5E+4 cells/well; about 1E+4 to about 4E+4 cells/well; about 1E+4 to about 3E+4 cells/well, about 2E4 to about 4E4 cells/well, or about 1E+4 to about 2E+4 cells/well. In some embodiments, cells are plated at a density of about 1E+4 , 2E+4 , 3E+4, or 4E+4 cells/well. In embodiments, these aforementioned cell densities are for a 96-well plate. In other embodiments, cells are plated at an equivalent density to these cell densities in a different size culture vessel, that would achieve similar confluence as these densities for a 96-well plate.

[0091] In some embodiments, cells are plated at a density of about 1E+4 +/- 50%; 1E+4 +/- 40%; 1E+4 +/- 30%; 1E+4 +/- 20%; 1E+4 +/- 10%; 1E+4 +/- 5%; 1E+4 +/- 1% cells/well. In some embodiments, cells are plated at a density of about 2E+4 +/- 50%; 2E+4 +/- 40%; 2E+4 +/- 30%; 2E+4 +/- 20%; 2E+4 +/- 10%; 2E+4 +/- 5%; or 2E+4 +/- 1% cells/well. In some embodiments, cells are plated at a density of about 3E+4 +/- 50%; 3E+4 +/- 40%; 3E+4 +/- 30%; 3E+4 +/- 20%; 3E+4 +/- 10%; 3E+4 +/- 5%; or 3E+4 +/- 1% cells/well. In some embodiments, cells are plated at a density of about 4E+4 +/- 50%; 4E+4 +/. 40%; 4E+4 +/- 30%; 4E+4 +/- 20%; 4E+4 +/- 10%; 4E+4 +/- 5%; or 4E+4 +/- 1% cells/well. In some embodiments, cells are plated at a density of about 2E4 to about 4E4 cells/well. In embodiments, these aforementioned cell densities are for a 96-well plate. In other embodiments, cells are plated at an equivalent density to these cell densities in a different size culture vessel, that would achieve similar confluence as these densities for a 96-well plate.

[0092] In some embodiments, cells are cultured subsequent to transduction with a viral vector. In some embodiments, cells are cultured for about 1, 2, 3, 4, or 5 days after transduction with a viral vector, e.g., prior to lysis. In embodiments, cells are cultured for about 1 day, about 2 days, about 1-4, 1-2, 1-3, 2-3, 2-4, 3-4, or 4-5 days after transduction, e.g., prior to lysis. In some embodiments, cells are cultured for about 2 days after transduction with a viral vector, e.g., prior to lysis.

[0093] In some embodiments, cells are cultured for between about 50-80 hours; 60- 80 hours, 60-70, 50-70 hours; 61-67 hours; 18-36 hours; 24-48 hours, 36-54 hours; 48-72 hours;36-48 hours; 38-52 hours, 40-58 hours; 44-66 hours; 40-50 hours; 42-49 hours, e.g., after transduction with a viral vector, e.g., prior to lysis. In some embodiments, cells are cultured about 60, 61, 62, 63, 64, 65, 66, 67, 68, or 69 hours, e.g., after infection, e.g., prior to lysis.

[0094] In some embodiments, cells transduced with a recombinant viral vector are lysed after being cultured. Those of skill in the art are aware of many methods to lyse cells. One of skill will also be aware that the mechanism of lysis needs to be compatible with any downstream use of the lysate (e.g., an expression assay or enzymatic activity-based assay). In some embodiments, a cell is lysed in a buffer lacking detergent. In some embodiments cells are lysed by freeze thaw cycling. In some embodiments, cells are lysed by at least 1, 2, 3 or 4, freeze thaw cycles. In some embodiments, cells are lysed in 80, 70, 60, 50, 40, 30μ l of buffer.

[0095] In some embodiments, a cell is lysed in a buffer comprising detergent. In some embodiments, cells are lysed by a buffer comprising Triton X-100; NP-40; deoxycholate; SDS; or TWEEN (e.g., about 0.05-0.1% TWEEN); or a combination thereof. In some embodiments, cells are lysed by a RIPA buffer (e.g., Sigma Aldrich R0278). In embodiments, cells are lysed in a buffer comprising a salt, e.g., at least 100 mM salt, e.g., at least 150 mM, 200 mM, 300 mM, 400 mM, or 500 mM salt, e.g., NaCl. [0096] In some embodiments a cell lysate is diluted. In some embodiments, a cell lysate is dilute prior to performance of an assay to determine activity of a protein in the lysate. . In some embodiments, a cell lysate is diluted prior to detection of expression of a protein in the lysate. In some embodiments a cell lysate is diluted in a buffer. In some embodiments, a cell lysate is diluted in a buffer to improve specificity and/ or sensitivity of an assay to determine activity or expression of a protein in the lysate. In some embodiments, a lysate is diluted in a buffer comprising one or more detergents. In some embodiments, a lysate is diluted in a buffer comprising Triton X-100; NP-40; deoxycholate; SDS; TWEEN (e.g., about 0.05-0.1% TWEEN); or a combination thereof. In embodiments, a lysate is diluted in a buffer comprising a salt (e.g., NaCl), e.g., at least 100 mM salt, e.g., at least 150 mM, 200 mM, 300 mM, 400 mM, or 500 mM salt, e.g., NaCl. In some embodiments, a lysate is diluted in RIPA buffer (e.g., Sigma Aldrich R0278), e.g., at a final concentration of about 50% to about 80% RIPA (1X-10X) and about 20-50% lysate. In some embodiments, a lysate is diluted in a buffer comprising PBS, 1% BSA, and 0.05% Tween 20. In some embodiments a lysate is diluted 1:2; 1:3; or 1:4. In embodiments, a lysate is diluted at least 2-fold, 3-fold, 4-fold, 5-fold, or more. Without wishing to be bound by theory, it is believed that the presence of detergent and/or salt in the dilution buffer can increase stringency in the detection step of an assay described herein, thereby reducing background.

Methods of Determining Biological Activity

[0097] Provided herein are methods for determining biological activity of a viral vector, e.g., recombinant viral vector. In some embodiments, determining the biological activity of a recombinant viral vector comprises determining the level of expression of a payload (e.g., a protein) encoded by the recombinant viral vector. In some embodiments, determining the biological activity of a recombinant viral vector comprises determining the activity (e.g., enzymatic) of a protein encoded by the recombinant viral vector. In some embodiments, activity can be determined, in whole or in part, by an enzymatic assay. In some embodiments, an assay is used to determine the function or activity of a protein encoded by a viral vector. In some embodiments, an assay is used to determine the activity of REP1, e.g., hREP1 encoded by a viral vector. In some embodiments, an assay, such as a prenylation assay, is used to determine the activity of REP1, e.g., hREP1.

Methods of Detecting Protein Expression

[0098] In some embodiments, level of expression of a protein encoded by a recombinant viral vector is determined from a cellular lysate. In some embodiments, the level of expression of a protein encoded by a recombinant viral vector may be carried out by any suitable method known in the art. In some embodiments, the level of expression can be determined by an enzyme-linked immunosorbent assay (ELISA, e.g, a sandwich ELISA), a Western blot, or autoradiography (e.g. utilizing an isotopically-labelled marker).

[0099] In some embodiments, the level of expression of a protein encoded by a recombinant viral vector is determined by an ELISA utilizing electrochemiluminescence. In some embodiments, the level of expression of a protein encoded by a recombinant viral vector is determined, in whole or in part, by a Meso Scale Discovery (MSD) based ELISA. The MSD platform uses an electrochemiluminescent technology to quantify signal. In some embodiments, a capture antibody is coated onto plates with electrodes in them, and a tag on the secondary antibody is activated by an electrical signal from the plate reader. This creates a luminescent signal that can be quantified and correlated with antigen level. The technology has been shown to have higher sensitivity, broader dynamic range, and lower background than a traditional ELISA method, which is based on colorimetric reaction of an HRP enzyme.

[0100] In some embodiments, a plate suitable for electrochemiluminescence is coated with an anti-Rep1 antibody (i.e., a capture antibody); Rep1 from the cellular lysate is then immobilized on the plate as a result of being bound by the anti-Rep1 capture antibody; the captured Rep1 may then be bound by a detection antibody. In some embodiments, the detection antibody comprises a label. In some embodiments, the detection antibody is unlabeled. In some embodiments, an unlabeled detection antibody is bound by a labeled secondary antibody. In some embodiments, a secondary antibody is specific for the species (e.g., mouse, rat, human, rabbit, or goat) of the detection antibody. In some embodiments a label is or comprises HRP or Ruthenium tris-bipyridine-(4-methylsufone) NHS Ester (e.g., a SULFO-Tag®). In some embodiments, the secondary antibody is SULFO-TAG® Anti- Rabbit Antibody Goat. Quantification of the label may be achieved by any suitable means (e.g. detection using a spectrophotometer, fluorometer or luminometer).

[0101] In some embodiments, level of expression of a protein encoded by a recombinant viral vector is determined relative to a standard. In some embodiments, relative level of expression is determined by parallel line analysis (PLA) against a standard curve, e.g., of a reference standard after linear regression data fit. In some embodiments, relative expression level is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, or higher relative to a reference standard. In embodiments, relative expression level is about 50% to about 150% relative to a reference standard.

[0102] Any suitable reference standard may be used. As used herein, a “reference standard” refers to a composition comprising a recombinant viral vector (e.g., encoding a REP1 polypeptide, e.g., human REP1 polypeptide), whose concentration and/or potency (e.g., expression level) is known.

Methods of Determining Protein Activity

[0103] In some embodiments, an activity assay comprises the steps of infecting cells with a viral vector encoding a protein of interest; culturing said cells; lysing said cells; and performing an assay to determine the enzymatic activity of a protein expressed in said cells that was encoded by the viral vector. In some embodiments, an assay to determine enzymatic activity is a prenylation assay. In some embodiments, a potency assay comprises a prenylation assay. In some embodiments, an enzymatic assay is a prenylation reaction reproduced in vitro to test for REP1 potency.

Prenylation

[0104] Previous methods for the detection of small GTPases in vitro use radiolabelled-prenyl donors. Radiolabelling can be replaced by either a fluorophore or a biotin group. Both approaches involve the use of a cultured cell lysate as REP1 is ubiquitously expressed in all cells and tissues. Protein incorporation of biotin-containing isoprenoids (biotin-labelled geranyl pyrophosphate, B-GPP) can be used to detect prenylated proteins due to their superior sensitivity relative to fluorescence-based methods.

[0105] Lipidation of proteins by the addition of isoprenoid moieties is a post- translational modification that affects up to 2% of the mammalian proteome. Such lipidation enables reversible association of the target proteins with cell membranes and can also modulate protein-protein interactions.

[0106] In some embodiments, lipidation referred to herein is prenylation. Prenylation is a specific type of post-translational modification in which a geranylgeranyl or farnesyl moiety (or analogue of either) is attached to one or two C-terminal cysteine residues of a protein via a thioether linkage.

[0107] In some embodiments, a lipid donor substrate and lipidated RAB6A product are a prenyl donor substrate and prenylated RAB6A product, respectively. In some embodiments, prenylation is the addition of a geranylgeranyl moiety or an analogue thereof (e.g. biotin-geranyl moiety) to a target protein (e.g. RAB6A).

[0108] A geranylgeranyl moiety attached to a protein (the protein is depicted schematically by the shaded circle) is:

[0109] A farnesyl moiety attached to a protein (the protein is depicted schematically by the shaded circle) is:

[0110] In some embodiments, an analogue of a lipid (e.g. geranylgeranyl or farnesyl) moiety or lipid donor substrate is used to determine potency of a vector encoded protein. In some embodiments, an analogue of a lipid is a lipid comprising a modification to comprise a functional group suitable for a particular purpose, e.g., detection. In some embodiments a lipid analogue is able to be added to a substrate protein by the prenylation machinery (i.e. REP1 and Rab GGTase) in a manner substantially unhindered (for the purposes of the activity assays described herein) by the modification.

[0111] In some embodiments, lipid analogues include those which have been artificially created for particular purposes (e.g. labelled moieties which are suitable for detection in an assay). For example, Nguyen et al. (Nguyen, U.T. et al. (2009) Nat. Chem. Biol. 5: 227-235) developed the following biotin-geranyl moiety that can be detected in in vitro protein prenylation reactions (the biotin-geranyl moiety is shown attached to a protein, which is depicted schematically by the shaded circle):

RAB6A

[0112] RAB6A (Ras-related protein Rab-6A) is a member of the mammalian Rab GTPase family, which is itself the largest of the Ras-like super-family of GTPases.

[0113] Rab GTPases (also known as Rab proteins) are peripheral membrane proteins and are involved in the regulation of membrane trafficking, including vesicle formation, vesicle movement along actin and tubulin networks, and membrane fusion. The main function of RAB 6 A is understood to be the regulation of protein transport from the Golgi complex to the endoplasmic reticulum.

[0114] Rab GTPases are typically anchored to a cell membrane via prenyl groups (in particular, geranylgeranyl groups) which are covalently bound to two C-terminal cysteine residues.

[0115] Rab GTPases exhibit two conformations: an inactive, GDP-bound form; and an active, GTP-bound form. Conversion from the GDP- to the GTP-bound forms is catalyzed by a GDP/GTP exchange factor (GEF), which thereby activates the Rab GTPase. Conversely, GTP hydrolysis by Rab GTPases can be enhanced by a GTPase-activating protein (GAP), which thereby leads to Rab inactivation.

[0116] In one embodiment, the RAB6A is human RAB6A.

[0117] An example amino acid sequence of RAB6A is the sequence deposited under NCBI Accession No. NP_942599.1 (SEQ ID NO: 7).

[0118] An example amino acid sequence of RAB 6 A is:

MSTGGDFGNPLRKFKLVFLGEQSVGKTSLITRFMYDSFDNTYQATIGIDFLSKTMYL EDRT VRLQLWDTAGQERFRSLIPSYIRDSTVAVWYDITNVNSFQQTTKWIDDVRTERGSDVI IM LVGNKTDLADKRQVSIEEGERKAKELNVMFIETSAKAGYNVKQLFRRVAAALPGMESTQD R

SREDMIDIKLEKPQEQPVSEGGCSC (SEQ ID NO: 8).

[0119] An example nucleotide sequence encoding RAB6A is the sequence deposited under NCBI Accession No. NM_198896.1 (SEQ ID NO: 9).

[0120] An example nucleotide sequence encoding RAB6A is:

ATGTCCACGGGCGGAGACTTCGGGAATCCGCTGAGGAAATTCAAGCTGGTGTTCCTG GGGG AGCAAAGCGTTGGAAAGACATCTTTGATCACCAGATTCATGTATGACAGTTTTGACAACA C CTATCAGGCAACAATTGGCATTGACTTTTTATCAAAAACTATGTACTTGGAGGATCGAAC A GTACGATTGCAATTATGGGACACAGCAGGTCAAGAGCGGTTCAGGAGCTTGATTCCTAGC T

ACATTCGTGACTCCACTGTGGCAGTTGTTGTTTATGATATCACAAATGTTAACTCAT TCCA GCAAACTACAAAGTGGATTGATGATGTCAGAACAGAAAGAGGAAGTGATGTTATCATCAT G CTAGTAGGAAATAAAACAGATCTTGCTGACAAGAGGCAAGTGTCAATTGAGGAGGGAGAG A GGAAAGCCAAAGAGCTGAATGTTATGTTTATTGAAACTAGTGCAAAAGCTGGATACAATG T

AAAGCAGCTCTTTCGACGTGTAGCAGCAGCTTTGCCGGGAATGGAAAGCACACAGGA CAGA AGCAGAGAAGATATGATTGACATAAAACTGGAAAAGCCTCAGGAGCAACCAGTCAGTGAA G GAGGCTGTTCCTGCTAA (SEQ ID NO: 10).

[0121] A further example nucleotide sequence encoding RAB 6 A is: gcacgcacgc acgcacgcca gcggccggcg gggccgcagg ctcgcgcccg ggctcgcccc 60 gcgccgctcc agaggctcgc gcactcagca ggttgggctg cggcggcggc ggcagctgtg 120 gaagctcagg cgctgcgcgt gagaggtccc agatacgtct gcggttccgg ctccgccacc 180 ctcagcttct cttccccagg tctgggagcc gagtgcggaa ggagggaacg gccctagctt 240 tgggaagcca gaggacaccc ctggctcctg ccgacaccgc cctccttccc ttcccagccg 300 cgggcctcgc tcggtgctag gctactctgc cgggaggcgg cggcggctgc cagtctgtgg 360 agagtcctgc tgccctccag ccgggctcct ccaccgggcc ttgcaggggc cgagagagct 420 cggtgcccgc ccttccgctc gcctttttcg tcagctggct ggagcagcat cggtccggga 480 ggtctctagg ctgaggcggc ggccgctcct ctagttccac aatgtccacg ggcggagact 540 tcgggaatcc gctgaggaaa ttcaagctgg tgttcctggg ggagcaaagc gttggaaaga 600 catctttgat caccagattc atgtatgaca gttttgacaa cacctatcag gcaacaattg 660 gcattgactt tttatcaaaa actatgtact tggaggatcg aacagtacga ttgcaattat 720 gggacacagc aggtcaagag cggttcagga gcttgattcc tagctacatt cgtgactcca 780 ctgtggcagt tgttgtttat gatatcacaa atgttaactc attccagcaa actacaaagt 840 ggattgatga tgtcagaaca gaaagaggaa gtgatgttat catcatgcta gtaggaaata 900 aaacagatct tgctgacaag aggcaagtgt caattgagga gggagagagg aaagccaaag 960 agctgaatgt tatgtttatt gaaactagtg caaaagctgg atacaatgta aagcagctct 1020 ttcgacgtgt agcagcagct ttgccgggaa tggaaagcac acaggacaga agcagagaag 1080 atatgattga cataaaactg gaaaagcctc aggagcaacc agtcagtgaa ggaggctgtt 1140 cctgctaatc tcccatgtca tcttcaacct tcttcagaag ctcactgctt tggccccctt 1200 actctttcat tgactgcagt gtgaatattg gcttgaacct tttcccttca gtaataacgt 1260 attgcaattc atcattgctg cctgtctcgt ggagatgatc tattagcttc acaagcacaa 1320 caaaagtcag tgtcttcatt atttatattt tacaaaaagc caaaatattt cagcatattc 1380 cagtgataac tttaaaaatt agatacattt tcttaacatt tttttctttt ttaatgttat 1440 gataatgtac ttcaaaatga tggaaatctc aacagtatga gtatggcttg gttaacgagc 1500 ggtatgttca cagcctactt tatctctcct tgcttttctc acctctcact tacccccatt 1560 ccctattacc ctattcttac ctagcctccc ccgacttcct caaaacaaac aagagatggc 1620 aaagcagcag ttctaccaag cccattggaa ttatccttta attttacaga taccacttgc 1680 tgtaggctac ggaccaagat gtccaaaatt attcttgagc actgatataa attacggtct 1740 tctttgaggt caaaattcag ccatcatggt aggcagtgct tgaatgagaa aaggctcctg 1800 gtgcatcttc aaaatgagtc ctaaagaaca tactgagtac ttagaagtag aagaacataa 1860 gatgtatttc tgactaaaac aaatggctct ttcacatgtg ctttattaga ctctgggaga 1920 gaaaattaac caagtgcttc agaacaggtt tttagtattt aattcttcac ggtaagaaaa 1980 tgaagttcta atgaactgtt tctcccaagg ttttaaaatt gtcaagagtt attctgtttg 2040 tttaaaaaat aagaaacctc tttaagcaat agattttgct tgggttttct tttttaaaaa 2100 cataatactg tgcaggcaag gcactgtaaa agttttaatt ccttccagaa gaaccagtgg 2160 aagaatttaa atttggcgct acgatcaaaa ctactgaatt agtagaaata atgatgtcta 2220 aagcttacca acaaaagaac cctcagcaga ataacaaaaa ctttgctcag gacatttgag 2280 gtcaaattga agacggaaac cggaaaccgt tttcttgtaa gcccctagag gcagatcagg 2340 taaagcatac atagtagagg gaaaggagag aatggaaata aaactcaata ttatgcagat 2400 ttatgcctta ttttttagca ttttttaagg ttgggtcttt caggctggtt ttggtttgta 2460 ttagatctgt atagtttaat taactggtga tttagtttta tatttaagct acaattaatc 2520 ttttttcttt ggtgatattt atttctttgc cttttttttt tttaacaact ttcaatcttc 2580 agatgtttcg ttgaatctat ttagagcttc accatggcaa tatgtatttc ccttaaaaca 2640 ctgcaaacaa atatactagg agtgtgccct tttaatcttt actagttatt gtgagattgc 2700 tgtgtaagct aataaacaca tttgtaaata cattgtttgc aggacgaaaa cttctgagtt 2760 acagctcagg aaaagcctgc tgaatttatg ttgtaagcat tacttaacac agtataaaga 2820 tgaaaagaca acaaaaatat cttcatactt cctcatcccc tcattggaac aaaaccttaa 2880 actgggagaa ccttagtccc ctctctttcc tcttcctcct ccacttccca cttattgtca 2940 ccttgtaata ttcagagagc acttggatta tggatctgaa tagagaaatg cttacagata 3000 atcattagcc cacataccag taacttatac ttaaagatgg gatggagttg taaagtgctt 3060 ttataataca atataattgt taaaggcaag ggttgactct ttgttttatt ttgacatggc 3120 atgtcctgaa ataaatattg attcaatatg gcagatgggt catattcttt atttggaaga 3180 agttgtgact tctgacatgg gtgtgattgt cttcctacac tgttgcattt gattcttttt 3240 atgtattttt aagaaagtaa ccagttatac tgcttttaat attgattggt ctttttattt 3300 ggcttggagt tcttcaaagc attgaagtgt gttcatagtc caggtttttt ttttaataaa 3360 cacaattttg ctgccaaaaa tatataaata aaacacgaaa gaaaacaaaa aaaaaaaaa (SEQ ID NO: 11) .

[0122] An example amino acid sequence of Rab27a is: 1 MSDGDYDYLI KFLALGDSGV GKTSVLYQYT DGKFNSKFIT TVGIDFREKR WYRASGPDG

61 ATGRGQRIHL QLWDTAGQER FRSLTTAFFR DAMGFLLLFD LTNEQSFLNV RNWISQLQMH

121 AYCENPDIVL CGNKSDLEDQ RVVKEEEAIA LAEKYGIPYF ETSAANGTNI SQAIEMLLDL

181 IMKRMERCVD KSWIPEGVVR SNGHASTDQL SEEKEKGACG C ( SEQ ID NO : 12 ) .

[0123] In some embodiments, a RAB6A as used for the methods or compositions described herein has 80%, 85%, 90%, 95%, 97%, 98%, or 99%, nucleic acid sequence identity to SEQ ID NO. 9, 10, or 11. In some embodiments, a RAB6A as used for the methods or compositions described herein has 80%, 85%, 90%, 95%, 97%, 98%, or 99%, amino acid sequence identity to SEQ ID NO. 7 or 8. In some embodiments, a RAB27A as used for the methods or compositions described herein has 80%, 85%, 90%, 95%, 97%, 98%, or 99%, amino acid sequence identity to SEQ ID NO. 12.

[0124]

Rab geranylgeranyltransferase (Rab GGTase)

[0125] Rab geranylgeranyltransferase (Rab GGTase; also known as geranylgeranyltransferase II) is a protein prenyltransferase which exclusively prenylates the GTPases of the Rab family.

[0126] Rab GGTase typically naturally catalyzes the transfer of two geranylgeranyl groups to cysteine residues at the C-terminus of Rab GTPases. Each geranylgeranyl group is conjugated to the Rab GTPase via a thioether linkage to a cysteine residue.

[0127] Rab GGTase has been shown to be capable of binding a range of derivatized phosphoisoprenoids and can catalyze their addition to Rab GTPase substrates (e.g. RAB6A). For example, Nguyen et al. (Nguyen, U.T. et al. (2009) Nat. Chem. Biol. 5: 227-235) demonstrated the successful addition of a biotin-geranyl moiety to Rab GTPases. [0128] Rab GGTase is a heterodimeric enzyme comprised of alpha and beta subunits.

[0129] In some embodiments, the Rab GGTase is human Rab GGTase. In some embodiments, the Rab GGTase is rat Rab GGTase.

[0130] Example amino acid sequences of Rab GGTase alpha subunits are the sequences deposited under NCBI Accession Nos. NP_004572.3 (SEQ ID NO: 13) and NP_113842.1 (SEQ ID NO: 14).

[0131] Example amino acid sequences of Rab GGTase alpha subunits are:

MHGRLKVKTSEEQAEAKRLEREQKLKLYQSATQAVFQKRQAGELDESVLELTSQILG ANPD FATLWNCRREVLQQLETQKSPEELAALVKAELGFLESCLRVNPKSYGTWHHRCLLGRLPE P NWTRELELCARFLEVDERNFHCWDYRRFVATQAAVPPAEELAFTDSLITRNFSNYSSWHY R SCLLPQLHPQPDSGPQGRLPEDVLLKELELVQNAFFTDPNDQSAWFYHRWLLGRADPQDA L RCLHVSRDEACLTVSFSRPLLVGSRMEILLLMVDDSPLIVEWRTPDGRNRPSHVWLCDLP A ASLNDQLPQHTFRVIWTAGDVQKECVLLKGRQEGWCRDSTTDEQLFRCELSVEKSTVLQS E LESCKELQELEPENKWCLLI ILLMRALDPLLYEKETLQYFQTLKAVDPMRATYLDDLRSKF LLENSVLKMEYAEVRVLHLAHKDLTVLCHLEQLLLVTHLDLSHNRLRTLPPALAALRCLE V LQASDNAIESLDGVTNLPRLQELLLCNNRLQQPAVLQPLASCPRLVLLNLQGNPLCQAVG I LEQLAELLPSVSSVLT (SEQ ID NO: 15) and:

MHGRLKVKTSEEQAEAKRLEREQKLKLYQSATQAVFQKRQAGELDESVLELTSQILG ANPD FATLWNCRREVLQHLETEKSPEESAALVKAELGFLESCLRVNPKSYGTHHRCWLLSRLPE P NWARELELCARFLEADERNFHCWDYRRFVAAQAAVAPAEELAFTDSLITRNFSNYSSHYR S CLLPQLHPQPDSGPQGRLPENVLLKELELVQNAFFTDPNDQSAWFYHRLLGRAEPHDVLC C VHVSREEACLSVCFSRPLTVGSRMGTLLLMVDEAPLSVEWRTPDGRNRPSHVWLCDLPAA S LNDQLPQHTFRVIWTGSDSQKECVLLKDRPECWCRDSATDEQLFRCELSVEKSTVLQSEL E SCKELQELEPENWCLLTI ILLMRALDPLLYEKETLQYFSTLKAVDPMRAAYLDDLRSKFLL ENSVLKMEYADVRVLHLAHKDLTVLCHLEQLLLVTHLDLSHNRLRALPPALAALRCLEVL Q ASDNALENVDGVANLPRLQELLLCNNRLQQSAAIQPLVSCPRLVLLNLQGNSLCQEEGIQ E RLAEMLPSVSSILT (SEQ ID NO: 16) and:

MHGRLKVKTSEEQAEAKRLEREQKLKLYQSATQAVFQKRQAGELDESVLELTSQILG ANPD FATLWNCRREVLQQLETQKSPEELAALVKAELGFLESCLRVNPKSYGTWHHRCWLLGRLP E PNWTRELELCARFLEVDERNFHCWDYRRFVATQAAVPPAEELAFTDSLITRNFSNYSSWH Y RSCLLPQLHPQPDSGPQGRLPEDVLLKELELVQNAFFTDPNDQSAWFYHRWLLGRADPQD A LRCLHVSRDEACLTVSFSRPLLVGSRMEILLLMVDDSPLIVEWRTPDGRNRPSHVWLCDL P AASLNDQLPQHTFRVIWTAGDVQKECVLLKGRQEGWCRDSTTDEQLFRCELSVEKSTVLQ S ELESCKELQELEPENKWCLLTI ILLMRALDPLLYEKETLQYFQTLKAVDPMRATYLDDLRS KFLLENSVLKMEYAEVRVLHLAHKDLTVLCHLEQLLLVTHLDLSHNRLRTLPPALAALRC L EVLQASDNAIESLDGVTNLPRLQELLLCNNRLQQPAVLQPLASCPRLVLLNLQGNPLCQA V GILEQLAELLPSVSSVLT (SEQ ID NO: 17) and:

MHGRLKVKTSEEQAEAKRLEREQKLKLYQSATQAVFQKRQAGELDESVLELTSQILG ANPD FATLWNCRREVLQHLETEKSPEESAALVKAELGFLESCLRVNPKSYGTWHHRCWLLSRLP E PNWARELELCARFLEADERNFHCWDYRRFVAAQAAVAPAEELAFTDSLITRNFSNYSSWH Y RSCLLPQLHPQPDSGPQGRLPENVLLKELELVQNAFFTDPNDQSAWFYHRWLLGRAEPHD V LCCVHVSREEACLSVCFSRPLTVGSRMGTLLLMVDEAPLSVEWRTPDGRNRPSHVWLCDL P AASLNDQLPQHTFRVIWTGSDSQKECVLLKDRPECWCRDSATDEQLFRCELSVEKSTVLQ S ELESCKELQELEPENKWCLLTI ILLMRALDPLLYEKETLQYFSTLKAVDPMRAAYLDDLRS KFLLENSVLKMEYADVRVLHLAHKDLTVLCHLEQLLLVTHLDLSHNRLRALPPALAALRC L EVLQASDNALENVDGVANLPRLQELLLCNNRLQQSAAIQPLVSCPRLVLLNLQGNSLCQE E GIQERLAEMLPSVSSILT ( SEQ ID NO : 18 ) .

[0132] Example amino acid sequences of Rab GGTase beta subunits are the sequences deposited under NCBI Accession Nos. NP_004573.2 (SEQ ID NO: 19) and NP_619715.1 (SEQ ID NO: 20).

[0133] Example amino acid sequences of Rab GGTase beta subunits are:

MGTPQKDVI IKSDAPDTLLLEKHADYIASYGSKKDDYEYCMSEYLRMSGIYWGLTVMDLMG QLHRMNREEILAFIKSCQHECGGISASIGHDPHLLYTLSAVQILTLYDS INVIDVNKWEY VKGLQKEDGSFAGDIWGEIDTRFSFCAVATLALLGKLDAINVEKAIEFVLSCMNFDGGFG C RPGSESHAGQIYCCTGFLAITSQLHQVNSDLLGWWLCERQLPSGGLNGRPEKLPDVCYSW W VLASLKI IGRLHWIDREKLRNFILACQDEETGGFADRPGDMVDPFHTLFGIAGLSLLGEEQ IKPVNPVFCMPEEVLQRVNVQPELVS (SEQ ID NO: 21) and:

MGTQQKDVTIKSDAPDTLLLEKHADYIASYGSKKDDYEYCMSEYLRMSGVYWGLTVM DLMG QLHRMNKEEILVFIKSCQHECGGVSASIGHDPHLLYTLSAVQILTLYDS IHVINVDKWAY VQSLQEDGSFAGDIGEIDTRFSFCAVATLALLGKLDAINVEKAIEFVLSCMNFDGGFGCR P GSESHAGQIYCCTGFLAITSQLHQVNSDLLGWWLCERQLPSGGLNGRPEKLPDVCYSWWV L ASLKI IGRLHIDREKLRSFILACQDEETGGFADRPGDMVDPFHTLFGIAGLSLLGEEQIKP VSPVFCMPEEVLQRVNVQPELVS (SEQ ID NO: 22) and:

MGTQQKDVTIKSDAPDTLLLEKHADYIASYGSKKDDYEYCMSEYLRMSGVYWGLTVM DLMG QLHRMNKEEILVFIKSCQHECGGVSASIGHDPHLLYTLSAVQILTLYDS IHVINVDKWAY VQSLQKEDGSFAGDIWGEIDTRFSFCAVATLALLGKLDAINVEKAIEFVLSCMNFDGGFG C RPGSESHAGQIYCCTGFLAITSQLHQVNSDLLGWWLCERQLPSGGLNGRPEKLPDVCYSW W VLASLKI IGRLHWIDREKLRSFILACQDEETGGFADRPGDMVDPFHTLFGIAGLSLLGEEQ IKPVSPVFCMPEEVLQRVNVQPELVS ( SEQ ID NO : 23 ) .

Lipid donor substrate

[0134] In some embodiments, to add a lipid moiety to a Rab GTPase, the Rab GGTase may use the lipid moiety in the form of a lipid (e.g. geranylgeranyl or biotin- geranyl) donor substrate as a substrate. In some embodiments, a donor substrate is a pyrophosphate derivatives of the lipid moiety.

[0135] In some embodiments, geranylgeranylpyrophosphate (GGPP) or biotin- geranylpyrophosphate (BGPP) may be used as lipid donor substrates by Rab GGTase to transfer a geranyigeranyl or biotin-geranyl moiety, respectively, to the substrate Rab GTPase. [0136] Geranylgeranylpyrophosphate has the structure:

[0137] An example structure of biotin- geranylpyrophosphate is:

Assay for Enzymatic Activity

[0138] In some embodiments a prenylation assay for determining the activity of REP1 comprises (a) providing a lysate comprising REP1; (b) contacting the lysate of step (a) with RAB6A, Rab geranylgeranyltransferase (Rab GGTase) and a lipid donor substrate; and (c) detecting a lipidated RAB 6 A product.

[0139] In some embodiments, a lipidated RAB 6 A product is a RAB 6 A to which a lipid moiety has been added. In some embodiments, the lipidated RAB 6 A product is a prenylated RAB6A, such as a geranylgeranylated RAB6A or a biotin-geranylated RAB6A.

[0140] Preferably, the RAB6A and/or Rab GGTase are from a standard source such that they provide for minimal or no variation in repeated experiments. Preferably, the RAB6A and/or Rab GGTase are substantially pure (i.e. comprise substantially no protein contaminants that interfere with the method or use of the invention).

[0141] In some embodiments, detecting the lipidated RAB 6 A product provides quantification of the amount of lipidated RAB6A product. The detection of lipidated RAB6A may be carried out by any suitable method known in the art. In some embodiments, a lipidated RAB6A product is detected by an enzyme-linked immunosorbent assay (ELISA, e.g., a sandwich ELISA), a Western blot, autoradiography (e.g. utilizing an isotopically- labelled, such as tritiated, lipid donor substrate), chromatographic (e.g. HPLC or FPLC) and/or mass spectrometry-based method (e.g. LC/MS). [0142] In some embodiments, a lipidated RAB6A product is detected by an ELISA utilizing electrochemiluminescence. In some embodiments, a lipidated RAB6A product is detected by a Meso Scale Discovery (MSD) based ELISA. The MSD platform uses an electrochemiluminescent technology to quantify signal. In some embodiments, a capture antibody is coated onto plates with electrodes in them, and a tag on the secondary antibody is activated by an electrical signal from the plate reader. This creates a luminescent signal that can be quantified and correlated with antigen level. The technology has been shown to have higher sensitivity, broader dynamic range, and lower background than traditional ELISA method, which is based on colorimetric reaction of an HRP enzyme.

[0143] In some embodiments, a prenylation reaction may be carried out according to the method of the invention using a biotin-geranylpyrophosphate lipid donor substrate. In some embodiments, a plate suitable for electrochemiluminescence is coated with an anti- Rab6a antibody (i.e., a capture antibody; see Figure 25); the product of a prenylation reaction is then immobilized on the plate as a result of being bound by the anti-Rab6a antibody; and then the lipidated RAB6A product (i.e. biotin-geranylated RAB6A) may be detected by a streptavidin based detection reagent. In some embodiments, a streptavidin based detection reagent is a streptavidin-SULFO tag or a streptavidin-horseradish peroxidase conjugate. Quantification of the lipidated RAB6A (i.e. biotin-geranylated RAB6A) may be achieved by any suitable means (e.g. detection using a spectrophotometer, fluorometer or luminometer).

[0144] In some embodiments, the method comprises a further step of comparing the amount of lipidated RAB6A product (e.g. prenylated, such as geranylgeranylated or biotin- geranylated, RAB6A) with an amount determined from a control experiment, such as an experiment using a known or standard sample of REPL In another embodiment, the method comprises a further step of comparing the amount of lipidated RAB6A product (e.g. prenylated, such as geranylgeranylated or biotin-geranylated, RAB6A) with a reference level.

[0145] The cell lysate comprising REP1 used in a prenylation assay may comprise about 1-20, 2-20, 1-10, 1-6, 2-5, 2.5-5.5; or 1.5-4.6 μg of total protein. In some embodiments, the cell lysate comprises 3.0, 3.5, 4.0, 4.5, or 5.0 μg of total protein. In embodiments, the cell lysate comprises at least about 2 to about 5 μg of total protein. In some embodiments the cell lysate protein quantity is about 5 μg. In some embodiments, the cell lysate protein quantity is at least about 1, 2, 3, 4, or 5 μg of total protein, e.g., about 2 ug or higher, e.g., about 2-20 ug, e.g., about 2 ug, 3 ug, 4 ug, 5 ug, 6 ug, 7 ug, 8 ug, 9 ug, 10 ug, 11 ug, 12 ug, 13 ug, 14 ug, 15 ug, 16 ug, 17 ug, 18 ug, 19 ug, or 20 ug. In some embodiments, 30, 20, 10, or 5 μl of cell lysate is used in the prenylation assay.

[0146] In some embodiments, a RAB6A in a prenylation assay is at a concentration of about 0.5 to 8 μM. In some embodiments, a RAB6A in a prenylation assay is at a concentration of about 0.1-10, 0.1-5, 1-10, 1-5, or 1-4 μM. In some embodiments, the a RAB6A in a prenylation assay is at a concentration of about, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM. In some embodiments, the RAB6A in a prenylation assay is at a concentration of 2 μM.

[0147] In some embodiments, a Rab GGTase enzyme in a prenylation assay is at least 0.5 μM. In some embodiments, a Rab GGTase enzyme in a prenylation assay is at a concentration of about 0.1-10, 0.1-5, 1-10, 1-5, or 1-4 μM. In some embodiments, a Rab GGTase enzyme in a prenylation assay is at a concentration of least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM. In some embodiments, the Rab GGTase enzyme in a prenylation assay is at a concentration of 1 μM. In some embodiments, a Rab GGTase enzyme in a prenylation assay is GGTase-II.

[0148] In some embodiments, a lipid donor substrate (e.g. biotin- geranylpyrophosphate (BGPP)) is at a concentration of about 1-25, 1-20, 1-15, 1-10 or 1-5 μM. In some embodiments, a lipid donor substrate is at a concentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 μM. In some embodiments, a lipid donor substrate is at a concentration of about 5 μM.

[0149] In some embodiments, a prenylation reaction may be carried out in any suitable buffer. In some embodiments, a prenylation reaction may be carried out in a prenylation buffer comprising about 50 mM HEPES, 50 mM NaCl, 2 mM MgC12, 1 mM DTT and protease inhibitor cocktail. In some embodiments a prenylation reaction buffer pH is about pH 7.1-7.5. In some embodiments a prenylation reaction buffer pH is about pH 7.2. [0150] In some embodiments, a prenylation reaction may be carried out for any suitable length of time at any suitable temperature (e.g. about 37°C). In some embodiments, a prenylation reaction may be carried out for about 1-10, 1-7.5, 1-5, 1-2.5 or 1-2 h. In some embodiments, a prenylation reaction may be carried out for about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 h. In some embodiments, a prenylation reaction may be carried out for preferably about 2 h at 37°C. In some embodiments, a prenylation reaction may be frozen (e.g., at -70°C) prior to detection of a lipidated RAB6A product.

Examples

Example 1: Expression Assay Development

[0151] The present example demonstrates development of a robust expression assay. The example describes reagents and processes to determine the expression of REP1 encoded by an AAV2 viral vector.

Cell Line Selection

[0152] HEK and Huh7 cells are known to be permissive for AAV2, and thus were selected to test for Rep1 expression. Two different HEK cell lines were used: the regular HEK293 and the transformed HEK293T cells. Infection was conducted at MOI=2.5E5 vg/cell (titer determined by qPCR) and Rep1 levels were analyzed by Western blot 3 days after infection. Infection and transduction are used interchangeably herein. As shown in Fig 1A, AAV infection significantly increased Rep1 level in HEK293 cells, however, a much higher expression was detected in HEK293T cells. Comparison of HEK293T and Huh7 cells were conducted by MSD methods. The MSD signal of HEK293T lysate was significantly higher than that from Huh7 lysate (Fig.1B). In conclusion, HEK293T produced the highest level of Rep1 after AAV infection.

MOIs and cellular response to rAAV2-Repl infection

[0153] AAV infection and the expression of transgenes may affect many biological activities of the host cells, including proliferation, morphology and survival, etc. It is important to make sure that host cells are healthy throughout the course of the experiment. HEK293T cells were infected by rAAV2-Rep1 at 2-fold serial dilution from top MOI = 1E6 vg/cell. As shown in Fig.2, under light microscopy, it was apparent that cell proliferation was significantly slowed down by infection at high MOIs (1E6-2.5E5 vg/cell (titer as determined by qPCR) and many cells showed a rounded morphology. The effect was much less prominent when infected at 1.25E5 vg/cell or lower. Based on this result, MOI of rAAV2-Rep1 infection that does not greatlyexceed 2.5E5 vg/cell is desirable.

Cell lysis buffer comparison

[0154] In order to detect a cytoplasmic protein like Rep1, cells must be broken by lysis buffer. Two different lysis buffers were tested: the milder (non-ionic) NETN buffer and the harsher RIPA buffer. In addition, NETN buffer was tested at 3 different strengths with 100, 300 or 500mM of NaCl. 3 days after infection at various MOIs, HEK293T cells were lysed by these 4 buffers at 4°C for 20min, and the amount of Rep1 was measured by MSD. As shown in Fig. 3, the levels of Rep1 detected in the experiments positively correlated to the harshness of the lysis buffer, with RIPA buffer yielding the highest signal in the assay. Without wishing to be bound by theory, this correlation is likely because Rep1 is an adaptor protein and may exist in protein complexes in mild lysis buffer, and is not accessible for immuno-detection.

Concentrations of capture and detection antibodies for immuno-detection

[0155] A monoclonal antibody produced in mouse was used as a capture antibody, and a polyclonal antibody from rabbit was used as a detection antibody. The concentration of monoclonal antibody was used as provided by the vendor, and an ELISA plate was coated at 3 concentrations: 4, 2 and 1 μg/mL. The concentration of the polyclonal antibody was not available, so it was tested at 100, 200 and 400-fold dilutions. Test samples were lysates of HEK293T cells infected at 3 different MOIs, and uninfected lysate was tested to determine background signal. As shown in Fig. 4, good signal to background ratio (> 6-fold) was achieved in all conditions tested. Considering the detection limits of ELISA, a suitable capture antibody concentration was determined to be 2 μg/mL and a suitable dilution factor for the detection antibody was determined to be 300-fold for the expression assay described herein.

Sample dilution for immuno-detection [0156] In this assay the test samples are whole cell lysates with many proteins other than Rep1. A low sample dilution is expected to increase immunoassay signal, but will also increase background due to matrix interference. To determine a proper dilution, lysates from cells infected at different MOIs were diluted from 4 to 32-fold in assay buffer for ELISA analysis. As shown in Fig. 5A, the shapes of the curves were similar for 4, 8 and 16-fold dilutions. The signal was significantly higher at low dilution, but the background stayed at about the same level. This observation was confirmed by repeating the experiment at 3 and 6-fold dilutions (Fig. 5B). In some embodiments, a 3-fold sample dilution was used for immuno-detection in the expression assay described herein.

Detection method comparison: ELISA vs MSD

[0157] The principles behind ELISA and MSD immunoassays are similar except for signal detection. While ELISA is based on colorimetric reading of an enzymatic reaction of the TMB substrate, MSD is based on electrochemiluminescent reading. See Figure 6. To compare these two detection methods, the same set of HEK293T lysate infected at 8 different MOIs were tested side by side by ELISA and MSD. Assay conditions optimized for ELISA, including capture and detection antibody concentrations and sample dilution were applied to MSD. As shown in Fig. 7, the high signal to low signal ratio of MSD was 16.4, compared to 6.5 by ELISA. Moreover, the ELISA curve started to inflect at the top 2 MOIs, while it stayed straight in the MSD curve. Without wishing to be bound by theory, this is likely because signal at the higher end approached the detection limit of ELISA. Based on these results, in some embodiments, MSD was used as the detection method for expression.

PLA models and the infection scheme

[0158] The MSD curve shown in Fig. 6 resembled a four-parameter logistic (4PL) curve without a well-defined upper asymptote, which was not reached because cells cannot stay healthy at a higher MOI (Fig. 2). Two different curve fits were tested: a 4PL fit that covers the whole curve and a linear fit that focuses on the slope. As listed in the table below, MOIs were adjusted according to the models used. In addition, rAAV2-Rep1 were tested at 50% and 150% strength of the MOI listed to assess assay accuracy.

[0159] As shown in Fig. 8, the relative potencies (RP) measured by both linear and 4PL model were within 80-120% of the expected values (50% and 150%), demonstrating good assay accuracy. However, the true linear range was very narrow as the ratio of high to low signal was only 2 fold. The range could not be extended to the lower end as the last dilution point of 50% RP fell out of the range and thus was excluded. In contrast, the 4PL fit with 8 point 2.5-fold serial dilution had a high to low signal ratio of 8.1-fold, a significant improvement over linear fit. The dilution scheme was further adjusted to reduce starting MOI and allow 6 points in the slope and 2 points in the lower asymptote (Uneven dilution). This significantly reduced AAV materials required per test, while maintaining assay performance such as accuracy, H/L ratios, etc. Notably, the assay has been demonstrated to be sensitive with a top MOI of 2.5E+05 or 3.66+05 followed by one 2-fold dilution, four 2.5-fold dilution and two 6-fold dilution (viral titer for MOI determined by qPCR).

[0160] Additionally, or alternatively dilutions can be prepared for the assay by preparing an initial dilution (e.g., top viral concentration) of 9.76E+7 Vg/ μl. Subsequently, one 2 fold dilution is prepared followed by four 2.5 fold dilutions; followed by two 6 fold dilutions. In some embodiments, 75 μl of each dilution is then added to cells in culture in 75 μl of media.

Conclusion

[0161] This assay has been developed to assess relative potency of rAAV2-Rep1 by quantifying relative levels of Rep1 protein expressed by infected HEK293T cells. The assay was developed through investigating antibody concentrations, cell lines, infection parameters, lysis buffer and data analysis models, and was optimized for the MSD detection platform. This assay is suitable for the support of process development, characterization, release, and stability studies where quantitative detection of Rep1 protein is needed.

Example 2: Potency Assay Development [0162] The present example demonstrates development of a robust potency assay.

The example describes the critical reagents and processes to determine the potency of REP1 encoded by an AAV2 viral vector.

Cell Line Selection

[0163] Choosing a cell line is the first step of successful development of a robust functional potency assay. An ideal cell line should easily uptake the viral vector and show a response that mirrors the mechanism of action of the drug; a strong high-to-low signal ratio and a fast response time are also important for a robust analytical assay. Original assay development was started with HEK293 cells, which have low levels of endogenous REP1 but show a visible increase of transgenic REP1 expression by Western blot following seven days of infection with rAAV2-hREP 1. Huh7 and HEK293T cells were chosen for further evaluation. These cell lines have both been shown to express a high level of protein of interest following AAV2 infection. Infection and transduction are used interchangeably herein. Surprisingly, expression of transgenic REP1 was significantly higher in HEK293T than in HEK293 (Figure 9).

[0164] To test whether Huh7 cells were a potential alternative, HEK293T and Huh7 cells were treated with AAV2-hREP 1 for 2 days. Cells were lysed and REP1 protein levels were assessed by MSD which showed lower levels of REP 1 in Huh7 compared to HEK293T (Figure 10). HEK293T cells were chosen for the next steps of assay development.

Infection Parameters

Optimization of Infection Time

[0165] Infection of the cells with rAAV2-hREPl was optimized to determine whether infecting cells for 2 or 3 days produces better curves or a higher ratio between the highest and lowest signal. As shown below in Figure 11, incubating cells for 3 days post- infection did not improve the amount of prenylated-Rab6a detected or the high-to-low signal ratio but extended the length of the assay. Infecting cells for 2 days was used for further development.

Optimization of Cell Seeding Density [0166] Some potency assays rely on seeding cells in 6-well plates. This design is only suitable for running a single MOI of a test article and does not allow for the setup of full dose-response curves necessary for a quantitative relative potency assay. For many reasons it is advantageous, to scale down the assay for use with 96-well plates. To determine the optimum cell density to use, cells were plated at three different densities per well. First, total protein extracted during lysis was evaluated. Six replicate wells of each density were lysed in 50 μL of prenylation buffer, and the total protein recovered was assessed with a Bradford assay (Figure 12A). The wells with 40,000 cells in them had higher protein extraction as well as less variability between wells.

Evaluation of the Infection Dilution Curve

[0167] Initially, to determine the optimum rAAV2-hREP1 MOI range for the REP1 relative functional potency assay, and to explore the asymptotes, infection was conducted with three different serial dilution schemes, each of 10-points. The first scheme was at a 1:1.5 dilution, the second was at a 1:2 dilution, and the third dilution scheme was split with 5 -points at a 1:2 dilution followed by 5 -points at a 1:3 dilution. While the lower asymptote was clearly observed in the second and third dilution schemes, a complete upper asymptote of the curve was never reached when plotting MSD counts against the log transformed value of MOI (Figure 13A). When double-log transformed, both the 1:1.5 and 1:2 dilution schemes were linear (respective R ² : 0.9953 and 0.9926) (Figure 13B). These experiments indicated that MOIs within this range could be selected for use in the final plate layout.

[0168] Early experiments evaluating a large MOI range, with titers determined by qPCR, indicated the general shape of the dilution curve. Because a strong linear portion of the curve was clear, a linear fit model was chosen for Parallel Line Analysis. A linear fit was chosen as opposed to a 4PL fit in order to conserve reagents and because we were not compromising on accuracy or precision with the linear fit. In order to determine the best 5- point dilution scheme for linear fit in PLA, three curves were tested with 50% and 150% samples. Scheme 1 and 2 both had a 1:2 dilution factor but had a top MOI of 5.0E4 and 2.5E4 VG/cell, respectively. Scheme 3 had a dilution factor of 1: 1.8 and top MOI of 3.0E4 VG/cell. As can be seen in Figure 13C, both the first and second schemes demonstrated a very slight hooking effect at either the top or bottom MOI, respectively, resulting in a non- parallelism with the control sample. Scheme 3, a tighter dilution scheme with a slightly lower top MOI fixed this problem and demonstrated great linearity.

Optimization of Lysis

Effect of Detergent on Downstream Prenylation Assay

[0169] Assay methods relying on manual cell lysis with a needle, e.g., one well at a time may be possible for use with a 6-well plate format, but are not feasible for a high- throughput assay, e.g., in a 96-well plate. One potential solution is to add detergent (Triton X-100 or NP-40) to efficiently lyse the cells. To test the effect of detergent on the prenylation reaction, various concentrations of Triton X-100 orNP-40 were mixed with an in vitro prenylation reaction using recombinant Rep1. As can be seen in Figures 14A-14C, the prenylation of Rab6a was significantly decreased in the presence of Triton X-100 or NP-40, even at 0.1 %. Based on these results, detergent could not be used during cell lysis as it interferes with the prenylation reaction.

Lysis by Freeze Thaw Cycling

[0170] Since the use of detergents in the cell lysis buffer was deleterious to the prenylation reaction, a freeze/thaw approach was implemented to lyse cells. HEK293T cells were seeded and two freeze/thaw cycles (-70°C/RT) were performed in prenylation buffer. The cells were monitored by microscopy to evaluate lysis. As can be seen in the microscopy images in Figures 15A-15C, the cells were efficiently detached and lysed after two freeze/thaw cycles.

[0171] After lysis by freeze/thaw cycling, total protein was quantified by a colorimetric protein assay kit and compared to cells lysed in NETN buffer to determine the extent of protein extraction. The protein quantitation, as seen in Figure 16A, demonstrated that the freeze/thaw approach efficiently extracted ~ 80% of the amount of protein extracted with NETN buffer. Freeze/thawing HEK293T cells allowed for effective and high throughput lysis without the use of detergents that interfere with the prenylation reaction.

[0172] In addition to showing that freeze/thaw cycling was sufficient to lyse the cells and extract protein, it was necessary to see whether the prenylation reaction could efficiently proceed with lysates generated with this method. To test this, HEK293T cells were treated with AAV2-hREP1 for two days, then medium was removed and replaced with prenylation buffer. The plate of cells underwent 0, 1, or 2 freeze/thaw cycles and lysates were used in a prenylation reaction. Prenylated Rab6a levels were assessed by MSD. As can be seen in Figure 16B, freeze/thaw cycling of the cells was sufficient to preserve functional Rep1 protein to use in the prenylation reaction. Although decent signal was seen after only one freeze/thaw, in one of the runs, signal was significantly lower than signal obtained with lysate that had undergone two freeze/thaw cycles. Two freeze/thaws were used in future experiments for consistency/robustness.

Use of a Shaker During Freeze Thaw

[0173] During assay development, it was thought that high frequency shaking on a specialized plate shaker (Micromix 5) would help homogenize the lysate and speed up the thaw cycles. To see whether this specialized equipment was necessary, an experiment was run where lysate was either thawed on the Micromix, on a standard shaker, or on the benchtop with no shaking. It was found that eliminating the shaking altogether was just as effective, simplified the protocol, and reduced the need for specialized equipment (Figure 17).

Effect of Lysate Centrifugation on Assay Performance

[0174] Even after freeze/thawing the lysate twice and pipetting up and down to mix, there were still visible clumps of cells floating in the wells (Figure 15A-15C). To reduce the risk of inconsistent results and well-to-well variability, a 10-minute centrifugation step was added to the protocol. As can be seen in Figure 18, centrifuging the lysate led to a slightly straighter line with tighter replicates and only a very small drop in signal.

Volume of Lysis Buffer

[0175] The efficiency of the prenylation reaction is dependent on the amount of total cell protein used. To optimize the reaction, different volumes of buffer were tried during lysis. Figure 19 shows that 50 μL of buffer led to better signal in the MSD reaction as compared to 80 μL of buffer, presumably because of the more concentrated Rep1 available in the lysate. Using less than 50 μL of buffer led to difficulty recovering the lysate after centrifugation. To efficiently recover the cell lysate and maximize signal in the MSD immunoassay, the cells were lysed in 50 μL of prenylation buffer.

Development of the in vitro Prenylation Reaction

[0176] Optimization of the in vitro prenylation reaction is important for effective potency measurement. It was necessary to evaluate the necessary amount of cell lysate, the final reaction concentration of both the substrate (Rab6a) and enzyme (GGTasell), prenylation reaction incubation time, and whether frozen lysate could be re-used for repeat assays.

Amount of cell lysate for the reaction

[0177] After establishing that a total lysis volume of 50 μL was to be used, two cell lysate volumes were identified - 10 and 19.5 μL - that would allow for at least two uses of the cell lysate but would allow for determining if lower amounts of protein were sufficient. These cell lysate volumes were measured by BCA for total protein extracted and contained 4.5 and 9 μg, respectively. As demonstrated (Figure 20), with 10 μL (4.5 μg) of total cell lysate, the MSD counts decreased by 30%. Nevertheless, the assay remained linear and performed well. In conclusion, the assay was further developed by using 10 μL (-4.5 μg) of cell lysate to reduce the amount of recombinant proteins (Rab6a and GGTase-II) used for the prenylation reaction. Protein is not measured within the assay but was investigated during development.

Optimization of the recombinant GGTase-II and Rab6a concentrations

[0178] Previous assays used the purified enzyme GGTase-II at a final concentration of 2 μM and the purified substrate, Rab6a, at a final concentration of 4 μM. To optimize the prenylation reaction and to better understand the overall kinetics of the reaction, various concentration of GGTase-II (1, 2, 4 μM) and Rab6a (1, 2, 4 μM) were tested. As expected, the signal increased with increasing concentration of GGTase-II enzyme and started to approach plateau (Figure 21A). For this type of assay the ideal enzyme concentration would be the one producing high enough signal but close to saturation, so small changes in concentration would not result in large signal variability, which can happen in the linear phase of the reaction. The second consideration was conserving the enzyme itself.

Therefore, a 1 μM final concentration of GGTase-II was chosen.

[0179] As observed in Figure 21B, increasing the concentration of Rab6a produced a bell-shaped curve with the highest signal at 2 μM (1 μM data not shown in MSD data, but in Western blot data the 1 μM band intensity was lower than 2 μM, hence the bell-shaped curve). This result was unexpected and indicated that concentrations above 2 μM inhibit the reaction efficiency, which suggests the presence of some inhibitory substance in the substrate preparation. We verified this phenomenon with two different substrate vendors and by Western blot. A 2 μM final concentration of Rab6a was chosen for further development.

Testing mixing for different length of the prenylation reaction

[0180] An initial prenylation reaction incubation time was set to 2 hours at 37°C without agitation. To better understand the robustness of this step and to potentially shorten the assay length, we evaluated both shorter incubation lengths (30 minutes, 1 and 2 hours) as well as the effect of agitation on reaction completion. Firstly, as demonstrated in Figure 22A, lowering the incubation time for the prenylation reaction significantly reduced the final MSD count. Secondly, as shown in Figure 22B, a 2-hour incubation with agitation was not significantly better (in terms of sensitivity; signal to noise ratio; or as variability/precision.) than without agitation, suggesting agitation is not required. In conclusion, the prenylation reaction performed for 2 hours at 37 °C without agitation achieved desired assay sensitivity.

Freezing prenylation reaction pre-MSD immunoassay

[0181] Experiments were performed to determine whether the infected, lysed, and frozen down cell lysate (at -70°C) can be thawed and re-used for prenylation reaction at a later date. This would increase assay flexibility and allow assay failures due to the prenylation reaction or MSD plate to be repeated without repeat infection. Experiments tested whether the assay could be stopped after the prenylation reaction by freezing the reaction at -70°C for one week and thawing at room temperature. As demonstrated (Figure 23), freezing the prenylation reaction did not affect the outcome of the potency assay. This frozen lysate was tested by two analysts, and it was demonstrated that there is little analyst variability. In conclusion, the assay can be frozen at -70°C after the prenylation reaction.

Development of Immunodetection Assay

Immunoassay Design

[0182] Immunoassays to detect prenylated products can rely on an ELISA format using a streptavidin coated plate for the immunodetection of biotinylated/prenylated Rab6a protein. A potential issue with this format is that the excess biotin GPP (B-GPP) in the biochemical prenylation reaction could interfere with the binding of prenylated Rab6a protein to the streptavidin coated plate. This was investigated by adding different concentrations of B-GPP to serial dilutions of purified recombinant biotin-Rab6a, and detecting with an anti-Rab6a primary Ab followed by a detection Ab. As demonstrated in Figure 24, increasing the amount of B-GPP significantly reduced the signal from purified biotin Rab6a. In conclusion, this ELISA assay format was determined not suitable for the functional potency assay and a new immunodetection format was developed.

[0183] In order to eliminate the effect of excess B-GPP in the prenylation reaction on the binding of biotinylated-Rab6a product, the sandwich-assay was redesigned (Figure25). The new design now begins by coating the plate with an anti-Rab6a antibody. The captured biotinylated Rab6a is then detected using a streptavidin-based detection reagent. As there was less un-biotinylated Rab6a in the reaction than free-floating B-GPP, this design resulted in less competitive interference.

Specificity of Rab6a Antibody

[0184] Antibody specificity is important for immunoassay performance. Two different monoclonal antibodies (SCBT, clones 38-TB and 3T3) and one polyclonal antibody (Abeam, Ab95954) recognizing Rab6a (not targeting the prenylated region of Rab6a) were evaluated. As shown in Figure 26, all the tested antibodies appeared to detect endogenous REP1 protein with similar sensitivity. When using the polyclonal antibody, the Rab6a band intensity appeared slightly darker, but the nonspecific higher band was also more intense. 38-TB had the faintest nonspecific band and was used in assays described herein.

[0185] Based on preliminary experiments, 2 μg/mL of 38-TB antibody was used for the coating concentration. The polyclonal antibodies under investigation showed much less sensitivity and had lower specificity.

Determining the optimal assay diluent

[0186] During development it was noted that when sample was loaded on the plate in wells with no capture antibody, the signal was higher than when there was capture antibody present. This observation suggests that the sample was sticking non- specific ally to the plate very effectively which is detrimental to assay specificity. To minimize this non- specific binding, the assay diluent (buffer in which the sample is diluted) was adjusted to be more stringent (inclusion of detergent). Experiments evaluating increased diluent stringency demonstrated an improvement in signal specificity. Initial assay diluent contained no detergent and resulted in more than double the signal when no capture antibody was used. With the addition of TBS-T-BSA (0.1% Tween, 1% BSA), the specificity increased dramatically, with more than 3x the signal in the presence of antibody as opposed to none. Further, by instead using RIPA , an even harsher detergent, we were able to improve not only specificity but also sensitivity and H:L signal (Figure 27). It was determined that the samples could be diluted in RIPA in methods described herein.

Coating time for capture antibody

[0187] Two different coating conditions were tested: A first plate and a second plate were coated 1 or 3 days in advance, respectively, and both were sealed and left in a fridge until use. 2 μg/mL of anti-Rab6a was used in both conditions, and cell lysate were produced from uninfected cells and cells infected by rAAV2-hREP 1 at 1.5E+5 VG/cell. The signal was similar in both coating conditions but the background signal (uninfected cells) doubled with a 3 days plate coating leading to a 2x decrease in the signal-to-background ratio. In some embodiments, a 1-day coating time was chosen to avoid unnecessarily high background signal. Wash buffer

[0188] The wash buffer, used between each of the MSD immunoassay steps, has a TBS base with 0.1 % Tween. Initially, the concentration for NaCl and Tris-HCl in the wash buffer differed from the other TBS containing buffers in the assay (coating buffer, blocking buffer, streptavidin detection buffer, etc.). Experiments were performed to evaluate whether this alternate recipe for TBS (137mM NaCl, 16mM Tris-HCl) was necessary or if it could be changed to other buffers (e.g., 150mM NaCl, 50mM Tris-HCl). It was found that there is no difference. Therefore, in the methods described herein, TBS based buffers can be made with 150mM NaCl and 50mM Tris-HCL.

Blocking buffer

[0189] Blocking buffer is useful for preventing non-specific binding of proteins to the plate, which improves the quality of the signal-to-background ratio. Experiments were performed to compare blocking buffers. Experiments compared a 1 % BSA/ PBS buffer to the SuperBlock blocking buffer (Thenno-Fisher), evaluated multiple BSA concentrations (1 %, 3%, and 5%) and looked at two different vendors (American Bio and Sigma Aldrich). The results show that the high-to-low signal ratio is higher with BSA as compared to superblock (H:L - 122, H:L - 50) and that neither increasing the percentage above 1 % or changing vendors made a difference. 1 % BSA was used in embodiments of the methods described herein.

Length of incubation on MSD plate

[0190] Incubation time of the prenylated reaction on the MSD plate was optimized to determine whether incubating the reaction for 1 or 2 hours produced similar curves or signal-to-background ratio. It was determined that incubating the prenylation reaction for 1 hour on the MSD plate was similar to the result obtained with a 2 hours incubation. To shorten assay length, incubating the reaction for 1 hour on the MSD plate was used in embodiments of the methods described herein.

Optimization of the detection reagent [0191] MSD technology has several advantages relative to standard ELISAs. MSD assays generally have an increased sensitivity, lower background, a high signal to background ratio, and a larger dynamic range. Another strong technical advantage is that output signal is not based on a time- sensitive reaction with TMB substrate. Upon switching to MSD technology, it was necessary to identify the optimal SULFOTAG labeled streptavidin dilution. 1 :500, 1: 1000 and 1 :2000 were compared. The detection reagent performed best at the 1:500 and 1: 1000 dilutions, which showed equivalent signal, while 1:2000 was slightly reduced. Therefore, in embodiments of the methods described herein, a 1: 1000 dilution could be used to conserve reagent while still detecting a strong signal.

[0192] Next, to further test specificity, purified biotin-Rab6a or biotin-RSI, diluted in either prenylation buffer or HEK.293T cell lysate was loaded onto the plate. As observed (Figure 28), biotin-Rab6a was efficiently detected in both conditions and the signal-to- background signal ratio was high. Furthermore, the detection of a separate biotinylated protein (B-RS1) was minimal and again demonstrated assay specificity.

Example 3- Expression Assay Procedure

[0193] The present example demonstrates an example procedure to determine the expression of REP1 encoded by an AAV2 viral vector.

Definitions and Abbreviations

Materials And Equipment Milli-Q or HPLC water may be used if WPU, Distilled, or RODI water is unavailable.

Solution Preparation

Complete Culture Medium for HEK293T Cells

[0194] Take a 50mL aliquot of FBS (heat inactivated) from -20°C freezer, and thaw in 37°C water bath. Add 50mL FBS to 500mL DMEM High-Glu Pyruvate medium. Mix by swirling the medium bottle 3 times and store in 4°C fridge protected from light. Assign expiration date of 1 month. All solutions below may be scaled based on assay needs.

20x PBS Stock Solution

[0195] Use a scale to weigh 160g NaCL, 43.2g Na ₂ HPO ₄ • 7H ₂ O, 4g KCL, and 4g KH ₂ PO ₄ . Dissolve dry chemicals in 800mL of distilled H ₂ O with magnetic stir. Adjust volume to IL by adding distilled H ₂ O. Store at room temperature for 6 months.

25% Tween 20 Stock Solution

[0196] Use a graduated cylinder to measure 250mL of Tween 20 and 750mL of distilled H ₂ O. Mix with magnetic stir and store at room temperature for 1 month.

Blocking buffer (IX PBS, 1% BSA) [0197] Use a scale to weigh 10g BSA, and dissolve in 800mL of distilled H ₂ O. Add 50mL of 20x PBS Stock Solution. Adjust volume to IL by adding distilled H ₂ O and with magnetic stir. Store at 2-8°C for 1 month.

Wash buffer (IX PBS, 0.05% Tween 20)

[0198] Transfer 500mL of 20x PBS Stock Solution to a IL graduated cylinder and add 9000mL of distilled H ₂ O. Add 20-mL of 25% Tween 20 Stock Solution or 5.0-mL of undiluted Tween-20. Adjust volume to 10 L by adding distilled H ₂ O. Mix with magnetic stir. Store at room temperature for 1 month.

Assay buffer (1X PBS, 1% BSA, 0.05% Tween 20)

[0199] Use a scale to weigh 10g BSA, and dissolve in 800mL of distilled H ₂ O. Add 50mL of 20x PBS Stock Solution. Add 2mL of 25% Tween 20 Stock Solution. Adjust volume to IL by adding distilled H ₂ O. Mix with magnetic stir. Store at 2-8°C for 1 month.

10% Bleach Solution

[0200] Add 100 mL of Bleach to 900 mL of H ₂ O. Make fresh solution before use.

Preparation of HEK293T Cells for Infection (Day 1)

Thawing Cells

[0201] Cell work should be conducted in BSL-1 lab in biosafety cabinets. All cell work should be done with sterile containers. All cell culture waste must be soaked in a 10% bleach solution for a minimum of 15 minutes prior to disposal. Warm complete culture medium in a water bath at 37°C. Remove 1-2 vials of HEK293T cells from the working cell bank stored in liquid nitrogen (LN ₂ ) to seed a single plate with the cells. Thaw enough vials of cells to obtain a starting cell concentration of approximately 3.0x10 ⁵ cells/mL to seed a single assay plate. Swirl the vial in a 37°C water bath until only a small crystal of frozen cells remain. Transfer the contents of the vial into a 15 mL conical tube with 8 mL of cold culture medium. Rinse the cell vial with 1 mL of cold culture medium and transfer the rinsate into the 15 mL conical tube. Centrifuge the tube for 5 minutes at 250 x g. Pour off the supernatant, cap the tube and disperse cell pellet by tapping the bottom of the tube with index finger. Resuspend cells in 7 mL of fresh warmed culture medium.

Seeding Cells

[0202] Cell work should be conducted in BSL-1 lab in biosafety cabinets. All cell work should be done with sterile containers and microwell plates. All cell culture waste must be soaked in a 10% bleach solution for a minimum of 15 minutes prior to disposal. Dilute 0.25mL cell suspension in 0.75mL culture medium (1:4), and count cells with Vi-Cell XR or hemocytometer for viability and cell density. Record cell count, measured cell density, and % viability. Cell viability should be ≥ 90%. Final cell density is 4 times of the measured value (Live cells/mL). Final cell density should be ≥ 2.8x10 ⁵ cells/mL. Dilute cells with culture medium to 2.67E+5 cells/mL in the 15 mL conical tube. Mix by pipetting gently up and down 5 times with 10mL serological pipette. Make 9 mL for one 96-well plate or scale up as needed.

[0203]

[0204] volume of media (mL) = total volume needed (mL) — volume of cells (mL)

[0205] Transfer the cell suspension into a sterile reagent reservoir. Using a multi- channel pipette, add 75 μL/well of cell suspension to 96-well cell culture plate (e.g. Costar 3595). Dispense the cell suspension row by row. Culture cells for 6 ± 1 hours in a 37±1°C incubator with 5±1% CO ₂ .

Infection of cells by AAV-Repl (day I)

[0206] Viral work should be conducted in Gene Therapy or other BSL-2 lab in biosafety cabinets. All virus dilutions should be done in sterile non-stick tubes or micro well plates. All viral waste must be placed in a 10% bleach solution for a minimum of 15 minutes prior to disposal. Preparation of viral interim dilutions

Control

[0207] If independent qualified control material is not available use the appropriate reference standard as the control.

Preparation of Reference Standard, Control, and Samples

[0208] Take the corresponding vials of STD, CTL and samples from -70°C freezer and thaw on ice. Tap the vials 3 times and spin at 250 x g for 15 seconds. Keep vials on ice till use. Dilute standard, control and samples to 1.95E+8 Vg/μL in 1.5mL nonstick tubes with warm culture medium. Make 350 μL for each preparation. Pipet up and down 5 times to mix. For one assay, make separate preparations for the standard, control and each sample. Make serial dilutions if needed. Do not pipet less than 10 μL of the AAV-Rep1 product. Remaining AAV-Rep1 materials should be stored at -70°C freezer in the analyst personal box. Do not return to common stock.

Standard curve and assay control preparation

[0209] One test plate can accommodate two samples per run. A master dilution plate must be prepared. Using a sterile 300 μL 96-well round bottom dilution plate (e.g. Corning 3365 or 3799), prepare the 8-point reference standard, control, and sample curves in triplicates. Using a multi-channel pipette, add 100 μL/well of warm complete culture medium to all wells. Dispense the medium row by row using reverse pipetting and touching tips to the bottom of the wells.

[0210] Prepare the top concentration for the reference standard, control, and samples in dilution plate (Top concentration is 9.76E+7 Vg/μL). Add 100 μL of the interim dilution (prepared in section 9.1 at 1.95E+8 Vg/μL) into respective wells containing 100 μL of medium on row A (Total volume in each well of Row A is 200 μL). Mix by pipetting up and down five times. [0211] Prepare one 2-fold dilution by transferring 100 μL from row A to row B using a P200 multichannel pipette. Mix well, but gently by pipetting up and down five times. Be careful not to produce too many bubbles.

[0212] Prepare 42.5-fold serial dilutions from row B to row F using a P200 multichannel pipette. Mix well, but gently, between each dilution by pipetting up and down five times. Transfer 66.7 μL from the corresponding row to the next set of wells. Be careful not to produce too many bubbles. Continue diluting 1:2.5 to row F. Leave transferred volume in wells of row F and dispose of the tips.

[0213] With new pipette tips, prepare 2 6-fold serial dilutions from row F to row G and continue through row H using a multichannel pipette. Mix well, but gently, between each dilution by pipetting up and down five times. Transfer 20 μL to the next set of wells by reverse pipetting. Be careful not to produce too many bubbles.

[0214] An example plate layout and concentrations of STD, CTL, and SAMPLE in the dilution plate are shown in Table 9.2-1.

Table 9.2-1. Serial dilution of rAAV2-REP1 for infection.

Viral Transduction [0215] All virus and cell culture waste must be soaked in a 10% bleach solution for a minimum of 15 minutes prior to disposal. Using a multi-channel pipette, transfer 75 μL/well of reference standard, control and sample to the corresponding wells in the 96-well cell culture plate (cells should have been in culture for 6+lh by then). Starting from Row H without changing tips, add test articles slowly and directly to the cell culture media (the pipette tips should touch liquid) as shown in Table 9.3-1. Be careful not to disturb the cells. Tap the plate gently to mix the viral solution. Culture cells with AAV-Rep1 materials in a 37°C incubator with 5±1% CO ₂ in Gene Therapy or other BSL-2 laboratory for 61 to 67 hours (64 ± 3 hours).

Table 9.3-1. Plate layout for infection of HEK293T cells by rAAV2-REPl.

Detection Of Repl Expression by MSD

[0216] REP1 MSD assay takes one and a half days during the 4-day procedure: On day one of the method, dilute capture antibody (Mouse anti-hREP1) and coat MSD plate.

[0217] On day four, the immunoassay major steps include: blocking for 1 hour (preparation of cell lysate during this time), incubation of samples for 2 hours, detection antibody for 2 hours, secondary antibody for 1 hour, and plate read on the MSD reader.

Coat MSD plate with REP1 antibody (Day 1 ) [0218] Prepare a solution of capture Ab (Mouse anti-hREP1) in PBS at a working concentration of 2 μg/mL. For one plate, add 10.6 μL antibody to 5.3mL PBS and mix by pipetting. Transfer the solution into a reagent reservoir. Using a multi-channel pipette, add 50 μL/well of the antibody solution to MSD plate. Use reverse pipetting to improve accuracy. Cover the plate with an aluminum plate sealer and incubate at 2-8°C for 3 days.

Blocking MSD Plate (Day 4)

[0219] Wash the coated MSD plate 2X with 300 μL/well PBS-T using a plate washer. Tap/blot the plate on paper towel. Block the MSD plate. Transfer the blocking buffer into a reagent reservoir and use a multichannel pipette to add 300μL/well of blocking buffer to the MSD plate. Cover the plate with a lid or foil plate sealer. Incubate the plate at room temperature for 60 +10 min without mixing.

Repl cell lysate preparation (Day 4)

[0220] 64 ± 3 hours after infection with AAV-Rep1 materials, remove the cell culture plate from the incubator. Examine cells under a light microscope. The majority of cells should be adherent to the plate. Inhibition of cell proliferation and some rounding up are expected for cells infected at the top MOI

[0221] This step should be conducted while blocking MSD plate(s). In a biological safety cabinet, use a multichannel pipette to remove culture medium. Start from Row H without changing tips. Minimize the amount of residual media left in the plate wells but be careful not to disturb cell monolayers. All viral and cell waste must be placed in a 10% bleach solution for a minimum of 15 minutes prior to disposal. Use a multichannel pipette, add 150 μL RIPA lysis buffer (protease inhibitor added before use) per well to the entire plate. Pipetting up and down 5 times to resuspend and ensure full lysis of the cells. From Row H to Row A without changing pipette tips. Lyse cells at 4°C for 20 min and inspect cells on a microscope after incubation to ensure full cell lysis.

[0222] Use a multichannel pipette, transfer lysates from the culture plate wells into a 96 well V- bottom plate (e.g. Corning 3894). Seal the plate with aluminum sealant sticker and spin the plate at 2200 G for 3 min to pellet cell debris. Use a multichannel pipette, transfer the supernatant from the V-bottom plate to a 96- well PCR plate. Minimize the disturbance of pellet in the bottom of the plate wells during transfer of the supernatant. Samples can be immediately tested by MSD (recommended) or stored at -70°C freezer for future analysis.

Dilute REP1 cell lysate in assay buffer.

[0223] Using a multi-channel pipette, add 50 μL/well of assay buffer to all wells of a 96-well round bottom plate (e.g. 3365 or 3799). Dispense the buffer row by row using reverse pipetting and touching tips to the bottom of the wells. Use a multichannel pipette to transfer 25 μL of Rep1 cell lysate from the 96-well PCR plate to the corresponding wells of dilution plate to be diluted 3-fold in assay buffer. Use reverse pipetting and touch liquid surface to improve accuracy. Mix each row of the dilution plate by pipetting up and down 5 times with a multichannel pipette. No need to change tips if going from low to high concentration of samples (bottom to top of dilution plate).

Detection of REP1 by MSD (Day 4)

[0224] Dump blocking buffer from the MSD plate into a sink. Be sure no liquid remains before adding samples by tapping the plate on paper towels. Using a multichannel pipette, transfer 50μL/well of the diluted Rep1 cell lysate samples from the corresponding wells of the dilution plate to respective wells of the MSD plate. Start from Row H without changing tips. The plate layout is the same as infection plate layout (Table 9.3-1). Cover the plate with a lid or foil plate sealer. Incubate the plate on a plate shaker at low speed (e.g. 200 rpm) at 22-26°C for 2 hours (+10 min).

[0225] Prepare a solution of detection Ab (Rabbit anti-hREP1) by diluting the reagent 1:300 in assay buffer. Make 5.3 mL for one assay plate (17.7 μL of detection Ab). Mix by pipetting. Wash the MSD plate 4 times with 300μL/well with PBS-T with plate washer. Be sure no liquid remains in the MSD plate by tapping/blotting the plate on paper towel.

[0226] Transfer the detection Ab solution into a reagent reservoir. Using a multi- channel pipette, add 50μL/well of the detection antibody solution to the MSD plate. Dispense the solution row by row starting from row H (lower sample concentration). No need to change tips. Cover the plate with a lid or foil plate sealer. Incubate the plate on a plate shaker at low speed (e.g. 200 rpm) at 22-26°C for 2 hours (+10 min). Prepare a solution of secondary Ab (SULFO-TAG® Anti-Rabbit Antibody Goat) by diluting the reagent 1:1000 in assay buffer. Mix by pipetting.

[0227] Wash the MSD plate 4 times with 300μL/well with PBS-T with plate washer. Be sure no liquid remains in the MSD plate by tapping/blotting the plate on paper towel. Transfer the secondary Ab solution into a reagent reservoir. Using a multi-channel pipette, add 50μL/well of the antibody solution to MSD plate. Dispense the solution row by row starting from row H (lower sample concentration). Cover the plate with a black lid or foil plate sealer. Incubate the MSD plate on a plate shaker at 200 rpm at 22-26°C for 1 hour (+5 min).

[0228] Prepare a IX Read Buffer solution by adding 4-mL of MSD Read Buffer Gold with surfactants (4x) to 12-mL of UltraPure water. Mix by pipetting. Wash the MSD plate 4 times with 300μL/well with PBS-T with plate washer. Be sure no liquid remains in the MSD plate by tapping/blotting the plate on paper towel. Transfer the read buffer into a reagent reservoir. Using a multi-channel pipette, add 150μL/well Read Buffer to MSD plate. Dispense the solution row by row starting from row H (lowest sample concentration). No need to change tips. Read the plate on the MSD reader within 15 minutes of adding the 1X Read Buffer solution.

Assay Acceptance Criteria

[0229] One of the outlying replicates for a particular dilution may be removed if it interferes with curve fitting (limited to one point per standard, sample, and/or control). Out of the three replicates, only the one with the highest deviation from the mean can be excluded.

[0230] Average signal-to-background (~ratio of top signal to lower asymptote values) for STD should be > 3, otherwise assay is invalid. [0231] The result of the sample and control must pass the tests of regression (F-test 95%), linearity (F-test 99.5%) and parallelism (equivalence test - ratio of slopes: between 0.5 and 2) in PLA in order for the relative potency to be valid.

Evaluate PLA results against the following acceptance criteria:

[0232] Control curve: The Control relative confidence interval must fall within VO- 143 % according to the PLA report. The relative potency of the Control should be within 80- 125%.

[0233] Sample curve: The Sample relative confidence interval must fall within VO- 143 % according to the PLA report.

[0234] If Control or Sample curve fails the above acceptance criteria, check for outliers. One replicate may be masked between the control curves, and one replicate may be masked between the sample curves. Re-evaluate the curve after outliers are masked.

[0235] If control fails, the whole assay is invalid. If control and 1 sample pass all criteria, result of the passing sample can be reported, and the failing sample should be retested.

[0236] If the relative potency of a sample is below 50% RP the sample should be reported as below linear range with the calculated value in parenthesis for the information only .If the relative potency is above the assay linear range (>150% RP), consult the submitter to see if the sample will need to be diluted down to the linear range and re-tested. If needed, a complete repeat test is performed in which the sample is diluted to a target 100% relative potency. The relative potency value obtained for the sample should be multiplied by the dilution factor to get the actual sample relative potency.

Example 4- Potency Assay Procedure

[0237] The present example demonstrates an example procedure to determine the potency of REP1 encoded by an AAV2 viral vector. Material And Equipment

Reagent Preparation

Culture Medium (DMEM + 10% FBS)

[0238] Add 50 mL FBS to 450 mL DMEM. Filter through a 0.2 μ m bottle-top filter. Assign an expiration date of one month from date of preparation and store at 4°C.

Coating Antibody Buffer (TBS)

[0239] Filtered 1X TBS: 50 mM Tris-HCl, 150 mM NaCl, pH 7.4 should be prepared as follows: Weigh out 198.2 grams of RO/DI water into the glass 250mL bottle. Weigh out 1.38 grams of Tris Hydrochloride and pour into the glass 250mL bottle. Weigh out 1.750 grams of Sodium Chloride and pour into the glass 250mL bottle. Mix solution with a magnetic stir bar until dissolved (10-15 minutes). Take pH specification, it should be 7.4 +/- .1.

Wash Buffer

[0240] Wash buffer: 16 mM Tris-HCl, 137 mM NaCl, 0.1% Tween-20, pH 7.8 should be prepared as follows: Weigh out 9,900 grams of RO/DI water into the plastic 10L bottle. Weigh out 25.2 grams of Tris Hydrochloride and pour into the plastic 10L bottle. Weigh out 80.0 grams of Sodium Chloride and pour into the plastic 10L bottle. Pipette 10 mF of Tween-20 (very viscous) and add it to the 10L of solution. Mix solution with a magnetic stir bar until dissolved (10-15 minutes). Take pH specification, it should be 7.8 +/- .1. Store at ambient temperature. Assign an expiration date of 1 month after preparation.

Blocking Buffer (TBS + 1% BSA) [0241] Warm BSA to room temperature before using. Combine 5 grams of BSA with 500 mL of TBS. Scale amounts as needed. Use a magnetic stir bar to mix until dissolved, then filter. Store at 4°C. Assign an expiration date of 1 month after preparation.

Streptavidin Dilution Buffer (TBS + 1% BSA + 0.1% Tween-20)

[0242] Warm BSA to room temperature before using. Combine 5 grams of BSA with 500 mL of TBS and 500 μL of Tween-20. Scale amounts as needed. Use a magnetic stir bar to mix until dissolved, then filter. Store at 4°C. Assign an expiration date of 1 month after preparation.

Prenylation Buffer (50mM HEPES, pH 7.2; 50mM NaCl; 2mM MgCl ₂ )

[0243] Filtered prenylation buffer should be prepared as follows: Weigh out 246.2g of RO/DI water and pour into sterile 250mL glass bottle. Weigh out 731mg of NaCl and pour into the sterile 250mL solution. Weigh out 47.6mg of MgC12 and pour into the sterile 250mL solution. Weigh out 2.98g of HEPES and pour into the sterile 250mL solution. Mix solution with a magnetic stir bar until dissolved (10-15 minutes). Take pH specification. It should be 7.2 +/- .1. Store at 4°C. Assign an expiration date of 1 month after preparation.

Assay Procedure

[0244] Cell work should be conducted in BSL-2 lab in biosafety cabinets. All cell work should be done with sterile containers. All cell culture waste must be soaked in a 10% bleach solution for a minimum of 15 minutes prior to disposal.

Day 1 (Morning): Thaw and plate cells

[0245] Warm complete cell culture medium in the 37°C water bath for 30 minutes before use. It is recommended to take out enough medium for one experiment rather than to warm up the whole bottle. Remove a vial of HEK293T cells from the working cell bank stored in liquid nitrogen (LN ₂ ). [0246] Swirl the vial in a 37+0.5°C water bath until only a small crystal of frozen cells remains. Do not leave vial unattended. Rinse the vial into a 15-mL conical tube with Culture Medium for a total of 10 mL. Centrifuge at 250 x g for 5 minutes. Pour off the supernatant.

[0247] After the supernatant is removed, gently tap the tube to resuspend the pellet, add 6 mL of warmed media with serological pipette and pipette the cells up and down 10 times. Use 0.5mL of the cell suspension to count cells with either a hemocytometer or the Vi-Cell XR. Record cell count, measured cell density, and % viability. Cell viability should be ≥ 90%.

[0248] Using the cell count, dilute cells to 0.53 x 10 ⁶ cells/mL. Prepare 6.5 mL of the cell suspension for one plate or scale up as appropriate.

[0249]

[0250] volume of media (mL) = total volume needed (mL) — volume of cells (mL)

[0251] Using an automatic multichannel dispensing pipette or a manual multichannel pipette with reverse pipetting, add 75 μL of the cell suspension row by row to a cell culture 96- well plate following the layout below.

[0252] Add 150 μL of medium to all empty wells. Cover the plate. Culture cells for 6+1 hour at 37+1 °C and 5+1% CO ₂ .

Day 1 (Afternoon): Treatment of Cells with AAV2-hREPl Preparation of Interim Dilution

[0253] Warm a 15-mL conical tube of medium in the 37±1°C water bath for 30 minutes before infection. Put AAV2-hREP1 standard and samples on ice or 4°C cold block to thaw for 20 minutes before infection. One test plate can accommodate two samples per run. Calculate the amount of material (standard, control, and each sample) needed to make 100 μL at 7.3 x 10 ¹¹ Vg/mL. Use the following equation:

[0254]

[0255] volume of medium to use = 100 pL — volume of viral material

[0256] Add 100 μL of warm culture media in 1.5mL Protein LoBind tubes. From each separate preparation made for the standard, control, and each sample, remove the calculated volume of media corresponding to the final volume of viral material to add. Add the calculated amount of the material to the tubes. Pipet up and down 5 times to mix. This is the interim dilution.

Preparation of Dilution Plate

[0257] Use reverse pipetting with a multi-channel pipette to add 198 μL/well of complete culture medium to columns 2 and 7 (wells B2 to G2 and B7 to G7) of a 300 μL 96- well, round bottom dilution plate as described in the table below . Dispense the medium by touching tips to the bottom of the wells.

[0258] Use reverse pipetting with a multi-channel pipette to add 98 μL/well of complete culture medium to columns 3-6 (wells B3 to G6) and columns 8-11 (wells B8 to Gil) following the plate layout below. Dispense the medium by touching tips to the bottom side of the wells.

[0259] Culture media plate layout prior AAV2-hREP1 addition

[0260]

[0261] Transfer 22 μL/ well of the interim dilution to the corresponding wells in column 2 (B2 to G2) and 7 (B7 to G7). Refer to the plate layout in Figure 8.2-2 below. The final volume of each well in columns 2 and 7 will be 220 μL.

[0262] Serially dilute 1:1.8 by transferring 122 μL from column 2 to column 3 using a P200 multichannel pipette. Mix well but gently between each dilution by pipetting up and down five times before transferring 122 μL to the next set of wells. Be careful not to produce too many bubbles and carefully monitoring pipette volumes for uniformity.

Continue the dilution series by transferring 122 μL from column 3 to 4 and so on through column 6. Change tips and perform the same dilution series described in 8.2.2.4 from column 7 to 11.

[0263] The plate layout and concentrations of STD, CTL, SAMPLE #1 and SAMPLE #2 in the dilution plate are shown in the table below.

[0264] Dilution plate layout of rAAV2-hREP1 for infection.

[0265] Store the remaining viral solution in -70±10°C freezer. Keep track of freeze/thaws. Mark clearly.

Viral transduction with AAV2-REP1

[0266] Using a P200 multi-channel pipette, transfer 75 μL/well of culture medium to the negative controls in wells Bl to DI, so in the end the final volume of all wells is 150μL/well.

[0267] Using a P200 multi-channel pipette, transfer 75 μL/well of reference STD, CTL and SAMPLE dilutions to the corresponding wells into the 96-well cell culture plate. Starting from Row B, add slowly in order not to disturb the cells. Change tips in between each transfer of virus to the cells. Repeat the procedure described above from Row B to Row G.

[0268] Culture cells in a 37±1°C incubator with 5±1% CO ₂ in Gene Therapy laboratory for 40±2 hours.

Day 2: Coat MSD Plate with Capture Antibody

[0269] Dilute the capture antibody (SCBT, cat#sc-81913) to 2 μg/mL in TBS by adding 80 μL of antibody to 3920 μL of TBS. Mix by inversion. Scale-up accordingly. [0270] Use a multichannel pipette to add 50 μL/well to Rows B-G of a high binding MSD plate. Gently tap the plate to ensure the liquid covers the bottom of the wells. Seal the plate and incubate overnight at 4°C.

Day 3: Cell lysis

[0271] Before beginning lysis, remove all the components for lysis and prenylation from the freezer and thaw them on ice (Halt Protease Inhibitor Cocktail, GDP, hRab6a, RabGGTase, B-GPP, hREP1, and B-Rab6a). Prepare the lysis buffer by mixing 6 mL of Prenylation Buffer with 60 μL of Halt Protease Inhibitor (1X final concentration).

[0272] In a viral-approved biological safety cabinet, with a multichannel P200 pipette set to 155uL, carefully remove medium from all wells of the infection plate. Tilt the plate and gently draw up the cell culture media from the corner of the well to avoid disturbing the cells. Bleach waste.

[0273] Using a multichannel P200 pipette, gently add 50 μL/well of the Prenylation Buffer/Protease Inhibitor solution to the cell-containing wells. Freeze the plate by incubating the plate for 15+1 minute at -70°C.

[0274] While the plate is in the freezer, make sure the centrifuge temperature is set to 4°C.

[0275] Take the plate out of the freezer and set on benchtop for 20+1 minute at RT. Freeze the plate at -70°C for an additional 15+1 minute, then take it out thaw on the benchtop for 20 minutes again.

[0276] Using a P200 multichannel pipette, mix each well up and down 5 times and transfer 45 μL of the lysate to a 96- well clear V-bottom TC-treated cell culture plate. Centrifuge the plate for 10 minutes at 2,200 x g at 4°C.

[0277] Tilt the plate and carefully remove 35 μL of the supernatant from the comer of the well to avoid disturbing the membrane pellet. Transfer the supernatant to a 96 well PCR plate. [0278] Keep the plate on ice. At this step, the cell supernatant can be stored at -70°C for future use or used immediately for the prenylation reaction.

Prenylation Reaction

[0279] Prepare an intermediate dilution of GDP in a 1.5 mL LoBind tube by adding 5 μL of GDP to 995 μL of Prenylation Buffer (a 1:200 dilution). Mix thoroughly.

[0280] Prepare the recombinant REP1 (rREP1) prenylation reaction positive control (0.03 μM) as described in Table 8.5-1. Dilute with Prenylation Buffer and mix in 1.5 mL LoBind tubes. The positive control must be prepared fresh for each assay.

Table 8.5-1: Preparation of the prenylation reaction positive control (rREP1)

[0281] Prepare the prenylation reaction master mix for 66 wells and 10% overage using the calculated reagent volumes (see e.g., Table 8.5-2). Add the reagents in the order described in the table below into a 0.5mL LoBind tube.

Table 8.5-2: Final concentrations of reagents for the prenylation reaction master mix.

[0282] NOTE: Final concentration is based on a final reaction volume of 15 μL. Also, volumes above are based on stock concentrations of hRab6a and RabGGTase-II at 5.76mg/mL and 8.5mg/mL, respectively. Adjust accordingly.

[0283] Set-up the VIAFLO Single Channel 50 μL Electronic Pipette on Repetitive Dispense mode with the following parameters: Dispense: 5 μL, Count: 6; Pre-Dispense: 2 μL; Post-Dispense: 5 μL; Aspiration/Dispense Speed: 8. The pre- and post-dispense will be performed in the tube containing the master mix.

[0284] Use the VIAFEO to add 5 μL of the master mix (by column) into wells B1- Gll of a 96- well EoBind plate by column. Seal both the PCR plate with mastermix as well as the plate containing cell lysate with a MicroAmp® Clear Adhesive Film then briefly centrifuge the plate with a bench centrifuge.

[0285] Remove the PCR plate adhesive film and use a P20 multichannel pipette to add 10 μL of the prenylation reaction control (rREP1 dilution) to wells El, Fl, and Gl. Add 10 μL of cell lysate into the corresponding wells of the prenylation plate (B1, C1, D1 and all inner wells). Pipette the cell lysate up and down 3-4 times and change tips between each addition. Seal the PCR plate and briefly centrifuge the plate.

[0286] Incubate for 2 hours at 37°C in a thermal cycler. Set up the thermocycler to keep the plate indefinitely at 4°C after the 2 hours.

Table 8.5-1 Prenylation reaction plate layout.

MSD Assay

[0287] Do not let the MSD plate dry out between steps. Prepare all buffers and reagents before washing the plate so they are ready to be added immediately after wash buffer is removed.

[0288] After 50 minutes of prenylation reaction, unseal the MSD plate precoated with the monoclonal Rab6a antibody as described in section 8.3. Dump out the contents, wash with Wash Buffer (TBS + 0.1% Tween-20, prepared in 7.3) on the automatic plate washer (one set of 4 washes) and gently tap the plate on paper towel to remove excess of wash buffer. Add 300 μL/well of Blocking Buffer (TBS + 1% BSA, prepared in 7.4) to rows B-G. Seal the plate and incubate it for Ihour ± 10 minutes at 24°C with shaking at 400 RPM.

[0289] While the plate is blocking, take out the RIPA buffer to warm it up to room temperature.

[0290] 10 minutes before the blocking is finished, prepare the MSD plate positive control (2500 ng/mL Biotin-Rab6a, refer to Table 8.6-1). Dilute with Prenylation Buffer and mix in 1.5 mL LoBind tubes. The positive control must be prepared fresh for each assay.

Table 8.6-1: Preparation of the MSD positive control (B-Rab6a)

[0291] At the end of the blocking step, the prenylation reaction described in section 8.5 will be over - briefly centrifuge the plate with a bench centrifuge.

[0292] Add 40 μL of 1X RIPA buffer to wells B1-G11. Use the same tips but touch them to the side of the wells and be careful not to touch the samples. Seal the plate and briefly centrifuge the plate with a bench centrifuge to make sure that the whole volume is at the bottom of the plate. Dump out the blocking buffer from the MSD plate. Wash the plate with Wash Buffer on the automatic plate washer (one set of 4 washes) and gently tap the plate on paper towel to remove excess of wash buffer.

[0293] Use a multichannel pipette to pipette the prenylation reaction up and down 3- 5 times, then transfer 50 μL to the blocked and washed MSD plate. The plate layout is identical - transfer to the corresponding well of the MSD plate as described below in Table 8.6-1. Change tips between each row and avoid making bubbles.

Table 8.6-1: Final MSD plate layout.

1 2 3 4 5 6 7 8 9 10 11 rAAVZ-REPl

[0294] Add 50 μL of the diluted MSD positive control into wells B12, C12 and D12 of the MSD plate as shown above in Table 8.6-1. Seal the plate and incubate it for 1 hours ± 10 minutes at 24°C with shaking at 400 RPM. [0295] Dilute the Streptavidin SULFO-TAG to 1:1,000 by adding 5 μL of Streptavidin SULFO-TAG to 5 mL of Streptavidin Dilution Buffer (TBS + 1% BSA + 0.1%

Tween-20, prepared in 7.5). Mix by inversion.

[0296] Dump off the contents of the MSD plate and wash the plate with Wash

Buffer on the automatic plate washer (one set of 4 washes) and gently tap the plate on paper towel to remove excess of wash buffer.

[0297] Add 50 μL/well of the diluted Streptavidin SULFO-TAG to Rows B-G with a multichannel pipette. Seal the plate and incubate for 1 hour + 10 minutes at 24°C with shaking at 400 RPM.

[0298] Prepare diluted Read Buffer by adding 8 mL of MSD Read Buffer (4x) to 8 mL of UltraPure water (2X final).

[0299] Dump off the Streptavidin solution. Wash the plate with Wash Buffer on the automatic plate washer (one set of 4 washes) and gently tap the plate on paper towel to remove excess of wash buffer.

[0300] Add 150 μL/well of the diluted MSD Read Buffer to Rows B-G. Use reverse pipetting and avoid bubbles.

[0301] Read the plate with the MSD reader within 15 minutes of adding the Read Buffer. Analyze data using PLA software.

Data Evaluation and Assay Acceptance Criteria

[0302] The result of the assay for the sample or control must pass the tests of regression (F-test 95%), linearity (F-test, 99.5%), and parallelism (equivalence test: ratio of slopes, 0.75-1.28) in PLA in order for the relative potency to be valid.

[0303] The average signal for the first point of the standard must be ≥ 2 times the average signal of the last point of the standard. The relative confidence intervals of the control and sample must be within 80-125%. The average signal of the negative control line (cells only) must be lower than the average signal of the last point of the standard. [0304] If all the above criteria pass, potency values can be reported. Otherwise, check the average signal of the positive control lines (hREP1 and B-Rab6a) to troubleshoot.

[0305] If the hREP1 or B-Rab6a positive control line averages are not ≥ 2 times higher than the average signal of the negative control line, the frozen down cell lysate plate can be used to rerun the prenylation reaction and MSD plate.

[0306] If the hREP1 or B-Rab6a control line averages are ≥ 2 times higher than the average signal of the negative control line, and any other criteria fail, the assay must be rerun from the beginning.

[0307] The linear range of the assay is 50-150%. If the sample relative potency is below 50%, report as “<50%”. For development samples, calculated values below 50% can be added to the comments for informational purposes only. If the relative potency is above 150%, the sample can be diluted and retested.

[0308] One of the outlying replicates for a particular dilution may be removed if it interferes with curve fitting (limited to one point per standard, sample, and/or control). Out of the three replicates, only the one with the highest deviation from the mean can be excluded.

Example 5 Evaluation of REP1 detection by MSD in hTERT-RPE cells

[0309] hTERT-RPE cells (ATCC CRL-4000) are an immortalized human retinal epithelial cell line. In the present example, the hTERT RPE-1 cell line was evaluated to determine whether it was responsive to treatment with rAAV2-hREP1. As an increase in REP1 protein levels is directly related to the mechanism of action of rAAV2-hREP1 showing activity in a tissue-specific cell line provides confidence that the signal seen in the HEK293T cells is representative of the viral vector’s effect in patient tissues.

[0310] hTERT-RPE were plated at 1.5E+4 cells/well and treated the same day with a 7 -point serial dilution of rAAV2-hREP1 (5+E5 to 3.9E+3 VG/mL, 2-fold serial dilutions). After three days, the cells had become completely confluent, with a very smooth and elongated morphology (Fig. 29A and 29B). Cells were lysed in 50 μL of RIPA buffer, and lysates from three treatment conditions (2.5E5, 6.25E4, and 0 VG/mL) were analyzed for REP1 protein levels by Western blot (mouse anti-REP1 clone 2F1, Sigma cat. # MABN52). There was a clear increase in REP1 levels with rAAV2-hREP1 treatment, as can be seen in Figures 30A and 30B.

[0311] Since a significant increase in REP1 protein levels was seen by Western blot with this cell line, the remaining lysates were analyzed by REP1 expression assay with MSD readout. The results are shown in Figure 31.

[0312] The hTERT RPE-1 cell line showed a clear increase in REP1 protein levels with rAAV2-hREP1 treatment when assayed by MSD. However, signal background ratio between the highest MOI and the untreated cells was around 3 ; in the HEK293T cells in Figure 29, this ratio is over 9. Additionally, overall signal was much lower with the hTERT RPE-1 cells (24,000 counts at the highest MOI vs 320,000 in the HEK293T cells). At least because of the lower signal and poorer signal background ratio, this cell line does not appear as robust as HEK293T cells in this assay context; however, its measurable response to rAAV2-hREP1 provides promising support for the mechanism of action of the rAAV2- hREP1 vector.

Equivalents

[0313] 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 invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims: