Shank3 potency assay

Abstract

Aspects of the disclosure relate to assays to measure activity of SHANKS proteins, including mini SHANKS proteins delivered by gene therapy.

Description

SHANK3 POTENCY ASSAYREFERENCE TO SEQUENCE LISTING

[0001] The contents of the electronic sequence listing (38061001 OP 1. xml; Size: 71,317 bytes; and Date of Creation: April 8, 2025) is herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0002] The present disclosure relates to a cell-based assay for measuring the potency of recombinant AAV vectors comprising an artificial genome comprising a SHANK3 -encoding transgene or any transgene that encodes a functional SHANK3 protein, including, for example, a miniSHANK3 transgene encoding a miniSHANK3 protein. Such AAV vectors can be used in patients undergoing gene therapy for delivering polynucleotides encoding a miniSHANK3 protein to a subject who has a neurodevel opmental disorder.BACKGROUND

[0003] Deletions and / or mutations involving SHANK3 account for about 0.5-1% of all autism spectrum disorder (ASD) patients and about 2% of ASD patients with intellectual disability (ID). However, there is no effective treatment for ASD and / or ID. Several challenges have arisen to developing pharmacological treatments that could correct the multitude of pathologies associated with ASD and ID. Gene therapy comprising rAAV vectors comprising an artificial genome comprising a miniSHANK3 transgene may be a treatment for ASD associated with SHANK3 mutations. There is a need for methods to assay and assess potency of the AAV gene therapy therapeutics for delivery of SHANK3 proteins.SUMMARY

[0004] Aspects of the disclosure relate to the development of assays to measure SHANK3 activity (binding to SHANK3 associated protein), including SHANK3 activity in gene therapy products, including AAV gene therapy products. Disclosed are methods for quantitating the activity of SHANK3 functional proteins, including miniSHANK3 binding activity, comprising: a) seeding cells onto a cell culture plate, wherein the cells contain a deletion of SHANK3 which obviates or negates SHANK3 activity in the cell; b) contacting the cells with a samplecomprising an AAV vector for a period sufficient for the AAV vector to transduce the cells, wherein the AAV vector comprises an artificial genome comprising a gene encoding a functional SHANK3 protein, including a miniSHANK3 transgene; c) lysing the cells to form a lysate; d) contacting the lysate to a substrate, wherein the substrate comprises a rGKAP, SAPAP, GRIP or Homer peptide that binds to SHANK3 or a SHANK3 associated protein; and e) measuring binding activity of SHANK3, miniSHANK3, or SHANK3 associated proteins to the rGKAP, SAPAP, GRIP or Homer peptide substrate, for example, by detecting the presence of SHANK3 or Homer bound to the substrate. Potency of a nucleic acid encoding SHANK3 proteins, including miniSHANK3 proteins, such as a miniSHANK3 AAV gene therapy therapeutic, may be assessed in the assay method disclosed herein, including by comparison to activity read outs from serial dilutions of a reference standard.EMBODIMENTS

[0005] Embodiment 1. A method for quantitating binding activity of a SHANK3 protein, including a miniSHANK3 protein, comprising: a) seeding cells onto a cell culture plate, wherein the cells contain a deletion of SHANK3 such that the cells do not have SHANK3 activity; b) contacting the cells with a sample comprising an AAV vector for a period sufficient for the AAV vector to transduce the cells, wherein the AAV vector comprises an artificial genome comprising a transgene encoding a SHANK3 protein, including SHANK3 or miniSHANK3; c) lysing the cells to form a lysate; d) contacting the lysate to a substrate, wherein the substrate comprises a peptide of a SHANK3 associated protein which binds to SHANK3 or a SHANK3 associated protein, such as rGKAP, SAPAP, GRIP or Homer peptide; and e) measuring binding activity of the SHANK3 protein, including, miniSHANK3,SHANK3 or SHANK3 associated proteins, to the rGKAP peptide attached to the substrate by detecting bound SHANK3 or SHANK3 associated protein, such as Homer. In an alternative, embodiment l is a method for quantitating binding activity of a SHANK3 protein, including a miniSHANK3 protein, comprising: a) seeding cells onto a cell culture plate, wherein the cells contain a deletion or inactivation of SHANK3 such that the cells do not have SHANK3 activity; b) contacting the cells with a sample comprising an AAV vector for a period sufficient for the AAV vector to transduce the cells, wherein the AAV vector comprises an artificial genome comprising a transgene encoding a SHANK3 protein, including a SHANK3 or a miniSHANK3 transgene; c) lysing the cells to forma lysate; d) contacting the lysate to a substrate, wherein the substrate comprises a SHANK associated protein such as GKAP, SAPAP, GRIP or Homer or a SHANK3 binding peptide thereof; and e) measuring binding activity of a SHANK3 protein or SHANK3 binding protein, including, miniSHANK3,SHANK3 or SHANK3 associated proteins, such as Homer, to the substrate.

[0006] Embodiment 2. The method of embodiment 1, wherein the substrate is rGKAP peptide which is conjugated to biotin.

[0007] Embodiment 3. The method of embodiment 2, wherein the rGKAP peptide is conjugated to biotin at the N-terminus by an SS linker.

[0008] Embodiment 4. The method of any one of embodiments 1 to 3, wherein the rGKAP peptide comprises NSATESAESIEIYIPEAQTRL (SEQ ID NO. 23).

[0009] Embodiment 5. The method of any one of embodiments 1 to 4, wherein the rGKAP peptide is Biotin - SS - NSATESAESIEIYIPEAQTRL (SEQ ID NO. 24).

[0010] Embodiment 6. The method of any one of embodiments 1 to 5, wherein measuring binding activity comprises (1) detecting bound SHANK3 or SHANK3 associated protein, such as Homer, to the rGKAP peptide with an anti-SHANK3 or anti- SAPAP, anti-GRIP or antiHomer antibody which recognizes the binding of SHANK3 or SHANK3 associated protein to the substrate rGKAP peptide; (2) detecting the anti-SHANK3 or anti- SAPAP, anti-GRIP or antiHomer antibody with a secondary antibody having a detectable label and (3) measuring a signal from the secondary antibody.

[0011] Embodiment 7. The method of embodiment 6, wherein presence of SHANK3 is detected and wherein the anti-SHANK3 antibody is Santa Cruz antibody SC-377470 or competes for binding to SHANK3 with the Santa Cruz antibody SC-377470.

[0012] Embodiment 8. The method of embodiment 7, wherein the secondary antibody is antimouse IgG HRP (ThermoFisher A16072).

[0013] Embodiment 9. The method of embodiment 6, wherein the anti-Homer antibody is Abeam ab97593 or competes for binding to Homer with Abeam ab97593.

[0014] Embodiment 10. The method of embodiment 9, wherein the secondary antibody is a goat anti-rabbit antibody, and in an embodiment may be IgG HRP (Thermofisher, #G21234).

[0015] Embodiment 11. The method of embodiment 9 or embodiment 10, wherein the Abeam ab97593 antibody is used at dilutions greater than 1 / 100.

[0016] Embodiment 12. The method of any one of embodiments 6 to 11, wherein the cells are seeded at a density of about le4 to about 4e4 per well and wherein the cells are contacted with the AAV vector in an amount of about lelO vector genomes (vg) to about 4el 1 vg per well.

[0017] Embodiment 13. The method of any one of embodiments 6 to 12, wherein the detectable label is a fluorophore.

[0018] Embodiment 14. The method of any one of embodiments 6 to 12, wherein the detectable label is an enzyme.

[0019] Embodiment 15. The method of embodiment 14, wherein the enzyme is horseradish peroxidase (HRP).

[0020] Embodiment 16. The method of any one of embodiments 12 to 15, wherein the signal is produced by interaction of the detectable label and a detector molecule.

[0021] Embodiment 17. The method of embodiment 16, wherein the detector molecule is 3,3-, 5 , 5 -Tetranm ethylbenzidine (TMB) .

[0022] Embodiment 18. The method of embodiment 16, wherein the detector molecule is QuantaBlu.

[0023] Embodiment 19. The method of any one of embodiments 2 to 11, wherein measuring binding activity of SHANK3 or SHANK3 associated proteins to the rGKAP peptide comprises measuring HRP activity with QuantaBlu.

[0024] Embodiment 20. The method of any one of embodiments 1 to 19, wherein the substrate is a microplate, including in embodiments a 96 well microplate.

[0025] Embodiment 21. The method of any one of embodiments 1 to 20, wherein the substrate is a neutravidin coated microplate, including in embodiments a 96 well microplate.

[0026] Embodiment 22. The method of any one of embodiments 1 to 21 wherein the substrate is a 96 well microplate and each microplate well is coated with at least 10 ng rGKAP peptide or the substrate is coated with an equivalent amount of rGKAP peptide proportional to the area of the microplate well.

[0027] Embodiment 23. The method of embodiment 22, wherein the substrate is a 96 well microplate and each well is coated with at least 100 ng rGKAP peptide or the substrate is coated with an equivalent amount of rGKAP peptide proportional to the area of the microplate well.

[0028] Embodiment 24. The method of any one of embodiments 1 to 23, wherein the cell culture lysate is in a CLBII buffer.

[0029] Embodiment 25. The method of any one of embodiments 1 to 24, wherein step b is performed in the presence of about 4 mM to about 40 mM hydroxyurea.

[0030] Embodiment 26. The method of any one of embodiments 1 to 25, wherein miniSHANK3 has an amino acid sequence of SEQ ID NO. 10 or SEQ ID NO: 12, or, in embodiments has an amino acid sequence having at least 90%, 95% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 12 and has SHANK3 activity or wherein the transgene comprises a nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or, in embodiments, comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95% or 99% identical to the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and encodes a functional miniSHANK3 protein.

[0031] Embodiment 27. The method of any one of embodiments 1 to 26, wherein the AAV vector is an AAV9.

[0032] Embodiment 28. The method of any one of embodiments 1 to 27, wherein the cells are human cells.

[0033] Embodiment 29. The method of any one of embodiments 1 to 28, wherein the cells are derived from U-87 MG cells.

[0034] Embodiment 30. The method of any one of embodiments 1 to 29, wherein the period sufficient for the AAV vector to transduce the cells is from about 48 to about 96 hours.

[0035] Embodiment 31. The method of embodiment 30, wherein the period sufficient for the AAV vector to transduce the cells is about 96 hours.

[0036] Embodiment 32. A method for quantitating SHANK3 protein activity, including activity provide by SHANK3, or miniSHANK3, comprising: a) seeding cells onto a cell culture plate, wherein the cells contain a deletion of SHANK3 such that the cells lack SHANK3 activity; b) contacting the cells with a sample comprising an AAV vector for a period sufficient for the AAV vector to transduce the cells, wherein the AAV vector comprises an artificial genome comprising a miniSHANK3 transgene; c) lysing the cells to form a lysate; d) contacting a neutravidin- coated microplate, including a 96 well microplate, with rGKAP peptides, wherein the rGKAP peptides are conjugated to a biotin at the N terminus with an SS linker, and wherein rGKAP peptides bind to the microplate to form a substrate; and e) washing the neutravidin-coated microplate; f) contacting the substrate rGKAP peptides with the lysate comprising SHANK3 or miniSHANK3; g) washing the neutravidin-coated microplate to remove unbound protein; h)contacting the biological sample with an anti-Homer antibody, wherein, in embodiments, the anti-Homer antibody is Abeam ab97593; i) washing the neutravi din-coated microplate; j) contacting the anti-Homer antibody with an HRP-conjugated secondary antibody; k) washing the neutravidin-coated microplate; 1) contacting the HRP-conjugated secondary antibody with QuantaBlu; and m) measuring the absorbance at 420nm.

[0037] Embodiment 33. A method for quantitating miniSHANK3 binding activity, comprising: a) seeding cells onto a cell culture plate, wherein the cells contain a deletion of SHANK3 such that the cells lack SHANK3 activity; b) contacting the cells with a sample comprising a viral vector, including a lentiviral vector, for a period sufficient for the AAV vector to transduce the cells, wherein the viral vector comprises an artificial genome comprising a miniSHANK3 or SHANK3 transgene; c) lysing the cells to form a lysate; d) contacting the lysate to a substrate, wherein the substrate comprises, or is, a GKAP, SAPAP, GRIP or Homer or a SHANK3 binding peptide thereof; and e) measuring binding activity of SHANK3, miniSHANK3 or SHANK3 associated proteins to the GKAP, SAPAP, GRIP or Homer or peptide thereof which binds to SHANK3 or a SHANK3 associated protein.

[0038] Embodiment 34. The method of embodiment 33, wherein the SHANK3 transgene encodes an amino acid sequence comprising SEQ ID NO: 6.

[0039] Embodiment 35. The method of embodiment 33 or 34, wherein the rGKAP peptide is conjugated to biotin.

[0040] Embodiment 36. The method of any one of embodiments 33 to 35, wherein the rGKAP peptide is conjugated to biotin at the N-terminus by an SS linker.

[0041] Embodiment 37. The method of any one of embodiments 33 to 36, wherein the rGKAP peptide comprises NSATESAESIEIYIPEAQTRL (SEQ ID NO: 23).

[0042] Embodiment 38. The method of any one of embodiments 33 to 37, wherein the rGKAP peptide is Biotin - SS - NSATESAESIEIYIPEAQTRL (SEQ ID NO: 24).

[0043] Embodiment 39. The method of any one of embodiments 33 to 38, wherein measuring binding activity comprises (1) detecting the binding of the SHANK3 or SHANK3 associated protein to the rGKAP peptide with an anti-SHANK3 or anti-Homer antibody which recognizes the binding of SHANK3 to the rGKAP peptide; (2) detecting the anti-SHANK3 or anti-Homer antibody with a secondary antibody having a detectable label; and (3) measuring a signal fromthe secondary antibody. In embodiments 33 to 34, binding may be detected using antibodies that bind to any one of the non-substrate SHANK3 associated peptides

[0044] Embodiment 40. The method of embodiment 39, wherein the anti-SHANK3 antibody is Santa Cruz antibody SC-377470 or competes for binding to SHANK3 with Santa Cruz antibody SC-377470.

[0045] Embodiment 41. The method of embodiment 40, wherein the secondary antibody is antimouse IgGHRP (ThermoFisher A16072).

[0046] Embodiment 42. The method of embodiment 39, wherein the anti-Homer antibody is Abeam ab97593 or competes for binding to Homer with Abeam ab97593.

[0047] Embodiment 43. The method of embodiment 42, wherein the secondary antibody is goat anti-rabbit IgG HRP (Thermofisher, #G21234).

[0048] Embodiment 44. The method of any one of embodiments 39 to 43, wherein the detectable label is a fluorophore.

[0049] Embodiment 45. The method of any one of embodiments 39 to 43, wherein the detectable label is an enzyme.

[0050] Embodiment 46. The method of embodiment 45, wherein the enzyme is horseradish peroxidase (HRP).

[0051] Embodiment 47. The method of any one of embodiments 39 to 43, wherein signal is produced by interaction of the signaling molecule and a detector molecule.

[0052] Embodiment 48. The method of embodiment 47, wherein the detector molecule is 3,3-, 5 , 5 -Tetranm ethylbenzidine (TMB) .

[0053] Embodiment 49. The method of embodiment 47, wherein the detector molecule is QuantaBlu.

[0054] Embodiment 50. The method of any one of embodiments 33 to 49, wherein the substrate is a microplate, including in embodiments a 96 well microplate.

[0055] Embodiment 51. The method of any one of embodiments 33 to 50, wherein the substrate is a neutravidin coated microplate.

[0056] Embodiment 52. The method of any one of embodiments 33 to 51, wherein the substrate is coated with at least 10 ng rGKAP peptide or the substrate is coated with an equivalent amount of rGKAP peptide proportional to the area of the microplate well.

[0057] Embodiment 53. The method of embodiment 52, wherein the substrate is coated with at least 100 ng rGKAP peptide or the substrate is coated with an equivalent amount of rGKAP peptide proportional to the area of the microplate well.

[0058] Embodiment 54. The method of any one of embodiments 33 to 53, wherein the cells are human cells.

[0059] Embodiment 55. The method of any one of embodiments 33 to 54, wherein the cells are derived from U-87 MG cells.

[0060] Embodiment 56. The method of any one of embodiments 1 to 55, wherein the method for quantitating miniSHANK3 binding activity has a precision (repeatability) with a percentage difference from identical samples of from about 5% to about 13%, from about 5% to about 12%, from about 5% to about 11%, from about 5% to about 10%, from about 5% to about 9% from about 5% to about 8%, or from about 5% to about 7%.

[0061] Embodiment 57. The method of any one of embodiments 1 to 56, wherein the method for quantitating miniSHANK3 binding activity is accurate and has a % recovery from about 110% to about 115%, from about 111% to about 114%, or from about 112% to about 113%.

[0062] Embodiment 58. The method of any one of embodiments 1 to 57, wherein the method for quantitating miniSHANK3 binding activity has an intermediate precision (%CV) of from about 1% to about 10%, about 2% to about 10%, about 3% to about 9%, about 4% to about 8%, or about 5% to about 7%.

[0063] Embodiment 59. The method of any one of embodiments 1 to 58, wherein the method for quantitating miniSHANK3 binding activity is linear and has an R2of about 0.98.

[0064] Embodiment 60. The method of any one of embodiments 1 to 59, wherein the method for quantitating miniSHANK3 binding activity has an adequate range of greater than equal to about 50% to less than or equal to 140%.

[0065] Embodiment 61. The method of any one of embodiments 1 to 60, wherein the method for quantitating miniSHANK3 binding activity is specific for mini-SHANK3 gene therapy and / or is able to detect changes in potency in AAV comprising mini-SHANK3 have been subjected to thermal stress.BRIEF DESCRIPTION OF THE DRAWINGS

[0066] FIG. 1 shows Western blot of SHANK3 expression in SHANK3 knock out cells (SKO) and U-87MG cells transfected with miniSHANK3 GOI (gene of interest) plasmid or vehicle control. Lanes 1 and 2 contain radioimmunoprecipitation assay (RIP A) generated lysates from mock transfected U-87MG and SKO cells, respectively. Lanes 3 and 4 contain RIPA generated lysates of miniSHANK3 GOI plasmid transfected SKO and U-87MG cells, respectively. Lanes 5 and 6 contain NP40 Cell Lysis Buffer generated lysates of miniSHANK3 GOI plasmid transfected SKO and U-87MG cells, respectively.

[0067] FIGs. 2A-B show the morphology of SKO cells transduced with plasmid encoding miniSHANK3 (FIG. 2B) or vehicle control (FIG. 2A).

[0068] FIG. 3 is a bar graph showing SHANK3 -specific absorbance for each binding / lysate condition shown.

[0069] FIG. 4 is a bar graph showing SHANK3 -specific absorbance for 0.1 pg / mL, 0.01 pg / mL or 0.001 pg / mL of rat or human GKAP peptide binding for 1 or 2 hours. Cells were lysed in either Cell Lysis Buffer II (CLBII) or RIPA Lysis Buffer (RIPA).

[0070] FIG. 5 is a bar graph showing SHANK3 -specific absorbance in lysates incubated for 1-3 hours using 0.1 pg / mL, 0.01 pg / mL or 0.001 pg / mL rGKAP (SEQ ID NO: 24) peptide coating concentrations.

[0071] FIG. 6 is a bar graph showing absorbance of SHANK3, SHANK3 interacting proteins, and secondary antibodies in vehicle and miniSHANK3-encoding plasmid transfected SKO lysates.

[0072] FIG. 7 is a bar graph showing absorbance of SHANK3 and SHANK3 -interacting molecules in vehicle and mini SHANK3 -encoding plasmid transfected SKO lysates incubated at room temperature or 37°C.

[0073] FIG. 8 is a bar graph showing SHANK3 and Homer-specific absorbance in vehicle and mini SHANK3 -encoding plasmid transfected SKO lysates.

[0074] FIG. 9 is a bar graph showing SHANK3 -specific and Homer-specific absorbance for each binding / lysate condition shown.

[0075] FIGs. 10A-B are line graphs showing SHANK3 -specific absorbance after 3 day (FIG. 10A) and 4 day (FIG. 10B) transductions of miniSHANK3 in SKO lysates plated at various densities with different concentrations of FBS.

[0076] FIGs. 11A-B are dot plot graphs showing SHANK3 -specific fluorescence in miniSHANK3 transduced SKO cells plated at various densities. QuantaBlu substrate was utilized to detect fluorescence, displayed as Log2RFU (relative fluorescence units) (FIG. 11 A) and SqRtRFU (FIG. 1 IB).

[0077] FIG. 12 is a bar graph showing SHANK3 -specific and Homer-specific fluorescence in vehicle and mini SHANK3 -encoding plasmid transfected SKO lysates screened for each binding / lysate condition shown.

[0078] FIGs. 13A-B are bar graphs showing SHANK3 -specific and Homer-specific fluorescence in vehicle, mini SHANK3 -encoding plasmid, and miniSHANK3 with Homer binding mutation-encoding plasmid transfected SKO lysates.

[0079] FIG. 14 is a line graph showing SHANK3 -specific fluorescence in miniSHANK3- encoding plasmid transfected SKO cells treated with various concentrations of hydroxyurea in SKO cell media.

[0080] FIG. 15 is a schematic showing the gene structure of full length SHANK3 and SHANK3 minigene.

[0081] FIG. 16 shows the linearity of individual test sessions A, B and C (observed % relative potency (RP) vs. expected % RP).

[0082] FIG. 17 shows the linearity of test sessions (observed % RP vs. expected % RP) from pooled data.

[0083] FIGs. 18A-B show dose response curves compared to JAG201 Reference Standard (circles). FIG. 18A shows SYN-RPL22 (squares) and FIG. 18B shows degraded JAG201 Pilot DS Mix (squares).DETAILED DESCRIPTION

[0084] Aspects of the disclosure relate to cell based methods for quantitating SHANK3 and binding activity of SHANK3, including miniSHANK3 and other functionally active forms of SHANK3 protein, including those expressed from a nucleic acid vector, such as an AAV gene therapy vector, binding activity to assess the potency of SHANK3 therapeutics, including nucleic acid vector SHANK3 (or mini-SHANK3) therapeutics, such as AAV gene therapy vectors,comprising: a) seeding cells onto a cell culture plate, wherein the cells contain a deletion of SHANK3, such that the cells lack SHANK3 activity, including SHANK3 binding activity, and are transducible by an AAV vector and in which the SHANK3 coding sequence is expressed; b) contacting the cells with a sample comprising the AAV vector for a period sufficient for the AAV vector to transduce the cells, wherein the AAV vector comprises an artificial genome comprising a mini SHANK3 -encoding or other SHANK3 -encoding transgene that is expressed in the cells; c) lysing the cells to form a lysate; d) contacting the lysate to a substrate, wherein the substrate comprises a rGKAP, SAPAP, GRIP or Homer peptide which binds to SHANK3 or SHANK3 associated protein; and e) measuring binding activity of SHANK3 or SHANK3 associated proteins, such as Homer directly, to the GKAP, SAPAP, GRIP or Homer protein or SHANK3 binding peptidethereof or via SHANK3 binding. In embodiments, potency can be assessed with comparing the binding activity measured according to the assay described herein to a reference standard. In embodiments where SHANK3 is detected by detecting binding to a SHANK3 associated peptide substrate such as rGKAP, SAPAP, GRIP, or Homer, such binding is detected with an antibody that binds to a non-substrate SHANK3 associated peptide, SHANK3 or miniSHANK3 protein.

[0085] SHANK3 encodes a synaptic scaffolding protein, which coordinates the recruitment of signaling molecules and orchestrates assembly of the macromolecular postsynaptic protein complex, which is crucial for proper synaptic development and function. Deletion of SHANK3 is a major cause of the core neurodevel opmental and neurobehavioral deficits in Phelan-McDermid syndrome. Human genetic studies also identified SHANK3 mutations as accounting for about 1% of autism spectrum disorder (ASD). Gene replacement is well suited as a therapeutic strategy for this monogenic disease.

[0086] Novel recombinant adeno-associated viruses (rAAVs) represent a promising gene delivery platform because of their wide range of tissue tropism, low immunogenicity, highly efficient and sustained gene transduction, and clinically proven track record in safety. However, it is known in the art that SHANK3 is a large protein with a coding sequence of about 5.7kb, exceeding the packaging capacity of AAV vectors. Miniaturized SHANK3 (“MiniSHANK3”) proteins described herein can be delivered by vector such as AAVs, including AAV9. AAV comprising a miniSHANK3 transgene can be used for treating neurodevelopmental disorders byrestoring the activity of SHANK3 in patients. Disclosed are assays used to compare and assess the potency of AAV compositions comprising a miniSHANK3 transgene.

[0087] In embodiments, disclosed are assays used to compare and assess potency of viral vectors, including viral vectors used for gene therapy, such as lentiviral vectors, adenoviral vectors or retroviral vectors, including adeno-associated virus (AAV) or nucleic acids (e.g., mRNA) comprising a miniSHANK3 or SHANK3 transgene.Potency Assays

[0088] A functional cell-based activity assay has been developed to demonstrate and quantitate potency of miniSHANK3 (or any protein with SHANK3 activity, such as wild type or full length SHANK3 or any variant thereof with SHANK3 activity), including nucleic acids encoding a SHANK3 or mini-SHANK3, gene therapy vectors that encode SHANK3 or a mini-SHANK3, including AAV vectors comprising a genome encoding a miniSHANK3 or nucleic acid vectors encoding SHANK3 or a miniSHANK3, via the SHANK3 -Homer 1 binding mechanism of action (or alternatively, through binding of SHANK3 and / or SHANK3 associated proteins to a binding partner). SHANK3 is a master scaffolding protein that binds and organizes scaffolding proteins at the synapses of excitatory neurons. Two such SHANK3 binding partners are Homer protein and guanylate kinase-associated protein (GKAP). Homer and GKAP, along with SAPAP and GRIP, are “SHANK3 associated proteins” in that they associate with SHANK3 in the cell at the scaffold and remain associated such that the presence of SHANK3 binding can be detected by detecting the presence of one of more of the SHANK3 associated proteins or SHANK3 itself. The disclosed cell-based assays assess and quantitate the functional binding activity of miniSHANK3 by detecting binding of miniSHANK3 to a GKAP peptide comprising the SHANK3 binding domain. If a complex between miniSHANK3 and GKAP forms, including complexes that include other SHANK3 associated proteins, such as Homerl, then the complex is retained on the substrate, detectable by an antibody against SHANK3 or an associated protein, including Homerl, and a secondary signal (for example, a labeled secondary antibody that recognizes the anti-SHANK3 or anti-SHANK3-associated protein, such as Homerl) is measured and used to assess the relative activity of the mini-SHANK3, including an AAV-miniSHANK3 gene therapy vector.

[0089] Aspects of the disclosure relate to methods for quantitating miniSHANK3 or SHANK3 binding activity, comprising: a) seeding cells onto a cell culture plate, wherein the cells contain adeletion or other mutation of the SHANK3 coding sequence causing a knock-out of SHANK3 activity; b) contacting the cells with a sample comprising an AAV vector for a period sufficient for the AAV vector to transduce the cells, wherein the AAV vector comprises an artificial genome comprising a miniSHANK3 or SHANK3 (or protein with SHANK3 activity) transgene operably linked to regulatory sequences that promote expression of the miniSHANK3 or SHANK3 protein in the cells; c) lysing the cells to form a lysate; d) contacting the lysate to a substrate, wherein the substrate comprises a rGKAP peptide; and e) measuring binding activity of SHANK3 or SHANK3 associated proteins to the rGKAP peptide, for example by detecting the presence of SHANK3 or a SHANK3 -associated protein, such as Homer, SAPAP, or GRIP bound to the rGKAP peptide (or other SHANK3 -binding peptide) bound to the substrate (such as a well of a 96 well plate).

[0090] The term “substrate” as used herein refers to any substance that is a solid support that is free of or substantially free of cellular toxins. In some embodiments, the solid substrate comprises one or a combination of silica, plastic, and metal. The substrate may have peptides bound to it, for example, through a biotin-avidin (or neuravidin) interaction where the substrate is covalently coated with the avidin or neuravidin.

[0091] The substrate may be in any form amenable to detecting binding to a rGKAP, SAPAP, GRIP or Homer peptide, which binds SHANK3 or to the scaffold complex containing SHANK3 through a SHANK3 associated protein, and to which the rGKAP, SAPAP, GRIP or Homer peptide can be attached or conjugated. In embodiments, the substrate is a microplate. A microplate, also known as a microtiter plate, microwell plate or multiwell, is a flat plate with multiple "wells" used as small test tubes. A microplate typically has 6, 12, 24, 48, 96, 384 or 1536 sample wells. In embodiments, the microplate is a 96 well microplate. Thus, amounts of rGKAP, SAPAP, GRIP or Homer peptide attached or conjugated to a well of a 96 well microplate may be extrapolated to wells of another size.

[0092] The GKAP, SAPAP, GRIP or Homer peptide may be attached or conjugated to the substrate using any method known in the art for attaching peptides to substrates. In embodiments, the GKAP peptide is conjugated to biotin and the substrate is coated with Neutravidin. In embodiments, the GKAP, SAPAP, GRIP or Homer peptide is conjugated to EDC and the substrate is coated with amino groups, e.g., Peptide Coating Kit (Takara Bio). In embodiments, the GKAP, SAPAP, GRIP or Homer peptide is conjugated to a His tag and thesubstrate is coated with either nickel or copper. In embodiments, the substrate is the well of a 96 well plate and about 10 ng, 50 ng, 100 ng GKAP, SAPAP, GRIP or Homer peptide (for example IOJJ.1, 50 pl, or lOOpl of a 0.1 pg / mL or 1.0 pg / mL solution of the GKAP peptide) is added to the well for conjugation. In embodiments, the amount of GKAP, SAPAP, GRIP or Homer peptide is proportional to the area of the substrate relative to the amount of GKAP, SAPAP, GRIP or Homer peptide and area of a 96 well plate.

[0093] In embodiments, the cell culture lysate is in a Cell Lysis Buffer II (CLBII). CLBII is 50 mM Tris, pH 7.4, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM NaiVO-t, 1% detergent, and 0.02% NaN3.

[0094] In embodiments, step b is performed in the presence of about 4 mM to about 40 mM hydroxyurea. In embodiments, step b is performed in the presence of about 10 mM to about 40 mM, about 15 mM to about 40 mM, about 20 mM to about 40 mM, about 25 mM to about 40 mM, about 30 mM to about 40 mM, or about 35 mM to about 40 mM hydroxyurea. In embodiments, step b is performed in the presence of about 40 mM hydroxyurea.

[0095] In embodiments, the period sufficient for the AAV vector to transduce the cells is about 24 to about 120 hours, about 36 hours to about 108 hours, or about 48 hours to about 96 hours.

[0096] In embodiments, the period sufficient for the AAV vector to transduce the cells is about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, about 108 hours, or about 120 hours.

[0097] In embodiments, the binding activity is detected with anti-SHANK3 or anti -Hom er antibodies that detect and bind to the SHANK3 (including mini-SHANK3)-Homer-GKAP complex formed in the assay and then, in embodiments, detectably labeled secondary antibodies that bind to the anti-SHANK3 or anti-Homer antibodies. Secondary antibodies are a population of immunoglobulins, including, for example, IgGs, generated in a host species that detect and bind to antibodies from a second species and can be detectably labeled. The secondary antibody detects antibodies of the species from which the anti-SHANK3 or anti-Homer antibody is derived. In embodiments, where the anti-SHANK3 or anti-Homer antibody is a rabbit antibody, the secondary antibody is a goat anti-rabbit antibody. In embodiments, where the anti-SHANK3 or anti-Homer antibody is a mouse antibody, the secondary antibody is a goat anti-mouse antibody.

[0098] In embodiments, the secondary antibody is directed against antibodies of the species of the primary antibody and is detectably labeled. In embodiments, the secondary antibody is Thermofisher, G21234 antibody (goat anti-rabbit HRP). In embodiments, the second antibody is ThermoFisher A-16035 antibody (donkey anti-rabbit IgG HRP). In embodiments, the second antibody is ThermoFisher Al 6072 (goat anti-mouse IgG HRP). Binding activity is detected and quantitated by measuring the detectable label, for example, fluorescence emitted from a fluorescent label or absorbance from other detectable markers as disclosed herein. Potency can be assessed and measured by comparison to a standard reference curve.Cells.

[0099] Cells used in the cell-based assay described herein can be any cell type that 1) expresses the proteins that associate with SHANK3 and 2) is transducible by the AAV therapeutic (or other SHANK3-based therapeutic) and 3) expresses the SHANK3 encoded by the therapeutic (i.e., the promoter that is operably linked to the SHANK3 coding sequence is active in the cells). The cells are engineered so that they do not express a SHANK3 protein that can participate in binding. In embodiments, the cells contain a deletion of SHANK3, i.e., are SHANK3 Knock-out cells (SKO) and / or do not otherwise have endogenous SHANK3 activity.

[0100] In embodiments, the cells used in the disclosed assay 1) express Homer protein; 2) are transducible by AAV (including the particular type of AAV of the therapeutic, including AAV9); and 3) must activate the promoter that drives expression of miniSHANK3 in the transgene construct within the AAV.

[0101] In embodiments, the SKO cells are generated from U-87 MG cells (ATCC, HTB- 14) a glioblastoma / astrocytoma cell line of human origin. U-87 MG cells are permissive to AAV9 transduction, drive expression of proteins from the synapsin promoter, and express the SHANK3 binding partner, Homer. In embodiments, the cells are human cells.

[0102] For the assay, the cells are seeded and cultured under appropriate cell culture conditions, for example, in wells of a 96 well plate. Media is appropriate for the cell line. In embodiments, the cells are seeded at a density of about le4, about 2e4, about 3e4, about e4 or about 5e4 cells per well on a 96 well plate. In embodiments, the cells are seeded at a density of about le4 to about 5e4 cells per well on a 96 well plate. For other cell culture formats, cells are seeded at a density that is proportional to the about le4 to about 5e4 cell number for the area of a well of a 96 well plate.

[0103] In embodiments, the cells are transduced with about lelO vg to about 4el 1 vg per well on a 96 well plate. In embodiments, the cells are seeded at 4e4 cells per well on a 96 well plate and transduced with 3el 1 vg per well. The amounts and ratio of cells to test article, e.g. vector genomes, depends upon the area and / or volume of the well or assay format and can be determined using methods known in the art. The amount and ratio of cells to test article will be standardized for any assay format, cell line and test article to permit assessment of activity relative to standards. miniSHANK3 AAV

[0104] In embodiments, the present disclosure provides methods of assessing the potency of a vector comprising a polynucleotide encoding a miniSHANK protein. In embodiments, the present disclosure provides a vector comprising a polynucleotide encoding a SHANK3 protein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is an AAV vector. In some embodiments, the present disclosure provides a recombinant AAV virion comprising a recombinant AAV genome and an AAV capsid. In embodiments, the present disclosure provides a recombinant AAV virion comprising an expression cassette encoding a miniSHANK3 protein under control of regulatory elements, such as a syn promoter, and an AAV9 capsid.

[0105] AAV refers to a replication-deficient (e.g., nonreplicating) Dependoparvovirus within the Parvoviridae genus of viruses. AAV can be derived from a naturally occurring virus or can be recombinant. AAV can be packaged into capsids, which can be derived from naturally occurring capsid proteins or recombinant capsid proteins. The single-stranded DNA genome of AAV includes inverted terminal repeat (ITRs). ITRs are involved in the replication and encapsidation of the AAV genome, along with its integration in the host genome and its excision. AAV vectors can comprise one or more ITRs, including a 5’ ITR and / or a 3’ ITR, one or more promoters, one or more nucleic acid sequences encoding one or more proteins of interest, and / or additional posttranscriptional regulator elements. In the present instance, the AAV vectors comprise recombinant genomes comprising a SHANK3 mini gene or SHANK3 -encoding transgene that produces the SHANK3 minigene protein product in target cells. AAV vectors disclosed herein can be prepared using standard molecular biology techniques known to one of ordinary skill in the art, as described, for example, in Sambrook et al. (Molecular Cloning: ALaboratory Manual. Cold Spring Harbor Laboratory Press, N.Y. (2012)), which is incorporated herein by reference in its entirety.

[0106] AAV vector sequences can be modified in any way known to one of ordinary skill in the art, such as by incorporating insertions, deletions or substitutions, and / or through the use of posttranscriptional regulatory elements, such as promoters, enhancers, and transcription and translation terminators, such as polyadenylation signals. In some embodiments, AAV vectors can include sequences related to replication and integration. In embodiments, the AAV vector comprises a nucleotide sequence of SEQ ID NO: 21.

[0107] AAV vectors can include any known AAV serotype, including, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11. In some embodiments, the AAV serotype is AAV9. Clades of AAV viruses are described in, and incorporated by reference, from Gao et al. (2004) J. Virol. 78(12):6381-6388. In some embodiments, any AAV serotype that is suitable for delivery to the CNS may be selected.

[0108] AAV vectors of the present disclosure may comprise or be derived from any natural or recombinant AAV serotype. In some embodiments, the AAV vector may utilize or be based on an AAV serotype described in WO 2017 / 201258A1, the contents of which are incorporated herein by reference in its entirety, such as, but not limited to, AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-lb, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43- 21, AAV43- 23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAVl-7 / rh.48, AAVl-8 / rh.49, AAV2-15 / rh.62, AAV2- 3 / rh.61, AAV2-4 / rh.5O, AAV2-5 / rh.51, AAV3.1 / hu.6, AAV3.1 / hu.9, AAV3-9 / rh.52, AAV3- 1 l / rh.53, AAV4-8 / rl 1.64, AAV4-9 / rh.54, AAV4-19 / rh.55, AAV5- 3 / rh.57, AAV5-22 / rh.58, AAV7.3 / hu.7, AAV16.8 / hu.l0, AAV16.12 / hu.l l, AAV29.3 / bb. l, AAV29.5 / bb.2, AAV106.1 / hu.37, AAV114.3 / hu.4O, AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54, AAV145.6Zhu.55, AAV161.1O / hu.6O, AAV161.6 / hu.61, AAV33.12 / hu.l7, AAV33.4 / hu.l5, AAV33.8 / hu.l6,AAV52 / hu.l9, AAV52.1 / hu.2O, AAV58.2 / hu.25, AAVA3.3, AAVA3.4, AAVA3.5, AAVA3.7, AAVC1, AAVC2, AAVC5, AAV-DJ, AAV-DJ8, AAVF3, AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70, AAVpi. l, AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVrh.69, AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVLK03, AAVH-l / hu.l, AAVH-5 / hu.3, AAVLG-10 / rh.40, AAVLG-4 / rh.38, AAVLG- 9 / hu.39, AAVN721-8 / rh.43, AAVCh.5, AAVCh.5Rl, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5Rl, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu.l, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9, AAVhu.10, AAVhu.l 1, AAVhu.13, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44Rl, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48Rl, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14 / 9, AAVhu.t 19, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.lO, AAVrh.12, AAVrh.13, AAVrh.l3R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.64, AAVrh.64Rl, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, AAAV, BAAV, caprine AAV, bovine AAV, AAVhEl.l, AAVhErl.5, AAVhER1.14, AAVhErl.8, AAVhErl.16, AAVhErl.18, AAVhErl.35, AAVhErl.7, AAVhErl.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1, AAV2.5T , AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV- PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC 12, AAV-2-pre-miRNA- 101 , AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2 ,AAV Shuffle 100-1 , AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8 , AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.l l, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, true type AAV (ttAAV), UPENN AAV 10, Japanese AAV 10 serotypes, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-El, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr- E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-Pl, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-Bl, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-Hl, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd-H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd-N9, AAV CLg-Fl, AAV CLg-F2, AAV CEg-F3, AAV CLg-F4, AAV CLg-F5, AAV CEg-F6, AAV CLg-F7, AAV CLg-F8, AAV CLv-1, AAV CLvl- 1, AAV Clvl-10, AAV CLvl-2, AAV CLv-12, AAV CLvl- 3, AAV CLv-13, AAV CLvl -4, AAV Civ 1-7, AAV Civ 1-8, AAV Civ 1-9, AAV CLv-2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-Dl, AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-El, AAV CLv-Kl, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-Ml, AAV CLv-Ml 1, AAV CLv-M2, AAV CLv-M5, AAV CLv-M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAV CLv-Rl, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-1, AAV CSp-10, AAV CSp-11, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8.10, AAV CSp- 8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1 / HSC1, AAVF11 / HSC11, AAVF12 / HSC12, AAVF13 / HSC13, AAVF14 / HSC14, AAVF15 / HSC15, AAVF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4, AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8,AAVF9 / HSC9, AAV-PHP.B (PHP.B), AAV-PHP.A (PHP. A), G2B-26, G2B-13, THE 1-32 and / or TH1.1-35, and variants thereof. AAV vectors are described further in US 9,585,971, US 2017 / 0166926, and W02020 / 160337, which are incorporated by reference herein in their entireties.

[0109] In embodiments, a MiniSHANK3 disclosed herein is delivered by a recombinant AAV vector. In embodiments, the recombinant AAV vector comprises a transgene encoding the MiniSHANK3 protein product and its regulatory sequences, and optionally 5' and 3' ITRs. In embodiments, the transgene and its regulatory sequences are flanked by the 5’ and 3’ ITR sequences. The transgene may comprise, as disclosed herein, one or more regions that encode a MiniSHANK3. The transgene may also comprise a region encoding for another protein. The transgene may also comprise one or more expression control sequences (e.g., promoter, a poly-A signal sequence). The transgene may be single stranded. In some embodiments, a recombinant AAV vector comprises at least AAV ITRs and a MiniSHANK3 transgene.

[0110] In some embodiments, the AAV may be packaged into an AAV particle and administered to a subject and / or delivered to a selected target cell. In some embodiments, the AAV particle comprises an AAV capsid protein. In some embodiments, the AAV particle comprises at least one capsid protein that is selected from the AAV serotypes as disclosed herein including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, PHB.eB, AAV.rh8, AAV.rhlO, AAV.rh39, AAV.43, AAV2 / 2-66, AAV2 / 2-84, and AAV2 / 2- 125, or a variant of any of the foregoing. In some embodiments, the AAV particle comprises an AAV9 capsid.

[0111] In embodiments, the AAV vector is an AAV9. In embodiments, miniSHANK3 has an amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 12 or has an amino acid sequence that is at least 85%, 90%, 95% or 99% identical to the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 12 and has SHANK3 activity.Shank3 binding peptides

[0112] As discussed above, SHANK3 is a master scaffolding protein that binds multiple proteins, including Homer and guanylate kinase-associated protein (GKAP). The SHANK3 binding domain of GKAP can be used to assess GKAP binding activity of miniSHANK3. Accordingly, peptides used in the assays described herein encompass the SHANK3 binding domain of the GKAP. In embodiments, the GKAP is the rat GKAP and has the amino acidsequence of SEQ ID NO: 22 in Table 1 below. In embodiments, the GKAP is the human GKAP and has the amino acid sequence of SEQ ID NO: 25 in Table 1 below.[001131 The SHANK3 binding domain of the rGKAP protein is within the 21 C-terminal amino acids of the protein and is underlined in SEQ ID NO: 23 in the table below. Accordingly, the peptide used in the assay comprises the SHANK3 binding domain of a GKAP protein, human or rat or other appropriate species. In embodiments, the rGKAP peptide comprises an amino acid sequence comprising NSATESAESIEIYIPEAQTRL (SEQ ID NO: 23). In other embodiments the GKAP peptide is a human GKAP peptide and has an amino acid sequence of SEQ ID NO: 26. In embodiments, the rGKAP peptide consists of the amino acid sequence of NSATESAESIEIYIPEAQTRL (SEQ ID NO: 23). In other embodiments the rGKAP peptide comprises NSATESAESIEIYIPEAQTRL (SEQ ID NO: 23) with one, two or three amino acid substitutions and the resulting modified peptide retains SHANK3 binding. In embodiments, the rGKAP peptide comprises the amino acid sequence of NSATESAESIEIYIPEAQTRL (SEQ ID NO: 23) and further comprises amino acid sequences from the rGKAP protein N-terminal of the C-terminal portion described such that the peptide is a 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid peptide portion of the rGKAP protein that contains the SHANK3 binding domain.

[0114] In alternative embodiments, the peptide may be a 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid peptide portion of SAPAP, GRIP or Homer that binds to SHANK3 or a SHANK3 associated protein.

[0115] The peptide is further conjugated to a moiety that mediates attachment to the substrate. The moiety may be directly attached to the N-terminus or C-terminus of the peptide or may be attached through a linker, including an amino acid linker or polyethylene glycol (PEG) or any other linker known in the art for attaching moieties to peptides. Examples of moieties that can be used to attach peptides to the substrate include biotin, when the substrate is coated is an avidin (e.g., avidin, neutravidin, streptavidin). Alternatively, the moiety is EDC and the substrate is coated with amino groups, e.g., Peptide Coating Kit (Takara Bio). In embodiments, the moiety is a His tag and the substrate is coated with either nickel or copper. In embodiments, the rGKAP peptide is conjugated to biotin. In embodiments, the rGKAP peptide is conjugated to biotin at the N-terminus by an SS linker. In embodiments, the rGKAP peptide is conjugated to biotin at the C-terminus by an SS linker. In embodiments, the rGKAP peptide is Biotin - SS -NSATESAESIEIYIPEAQTRL (SEQ ID NO: 24). In embodiments, the rGKAP peptide is Biotin -NSATESAESIEIYIPEAQTRL (SEQ ID NO: 23). Alternatively, the peptide is a human GKAP peptide conjugated to biotin directly or through an SS linker, including a peptide which as an amino acid sequence of SEQ ID NO: 27. The amount of the peptide added to the substrate depends upon the area of the substrate to be coated. In embodiments, the substrate is the well of a 96 well microplate and the amount of peptide added to the well for conjugation is 10 ng to 100 ng, or about 10 ng, about 50 ng or about 100 ng.

[0116] In embodiments, the rGKAP peptide is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to NSATESAESIEIYIPEAQTRL (SEQ ID NO: 23) and retains SHANK3 binding. The terms “sequence identity”, “percent sequence identity” or “percent identical” in the context of nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over the full-length of a gene sequence or component as provided in the sequence listing or claimed, e.g. an enhancer or promoter or composite promoter sequence, or a fragment or portion thereof, for example over a nucleotide sequence encoding a composite enhancer / ACTA core promoter. However, identity among smaller fragments, e.g. of at least about ten nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides, may also be desired. Similarly, “sequence identity”, “percent sequence identity” or “percent identical” may be readily determined for amino acid sequences, over the full-length of a protein, or a fragment or portion thereof, such as for transgene protein products or capsid proteins.

[0117] In embodiments, the peptide substrate can be a SHANK3 -binding peptide portion of a protein that binds SHANK3 (such as GKAP, SAPAP, GRIP or Homer, including human and non-human variations thereof).

[0118] Table 1. Sequences

[0119] In embodiments, other linkers, such as PEG, can be used to conjugate the rGKAP peptide to a binding moiety. In embodiments, glycine and serine linkers (Ser / Gly linkers) can be used to conjugate the rGKAP peptide to a binding moiety. In embodiments, there is no linker and the rGKAP peptide is conjugated directly to the binding moiety, e.g., biotin.

[0120] In embodiments, the rGKAP peptide is about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24 or about 25 amino acids long and binds SHANK3.

[0121] In embodiments, the GKAP peptide is the C-terminal 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids of rGKAP and binds SHANK3.

[0122] In embodiments, the rGKAP peptide has 1, 2, or 3 or more conservative modifications compared to the original rGKAP peptide, but is still capable of binding SHANK3. Conservative substitutions include the replacement of one amino acid residue with another that is biologically and / or chemically similar. For example, a conservative substitution would be replacing one hydrophobic residue for another, or one polar residue for another. The substitutions include combinations such as, for example, Gly, Ala; Vai, He, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variations of each explicitly disclosed sequence are included within the mosaic polypeptides provided herein.

[0123] In embodiments, the substrate is a 96 well plate and each well is coated with at least about 10 ng rGKAP peptide. In embodiments, the substrate is a 96 well plate and each well is coated with at least about 100 ng rGKAP peptide.

[0124] In embodiments, the substrate is a 96 well plate and each well is coated with about 10 ng to about 100 ng rGKAP peptide. In embodiments, for other assay formats, the surface of the assay format would be coated with an amount of peptide that is proportional to 10 ng to 100 ng of rGKAP peptide in the well of a 96 well plate.Antibody Detection System

[0125] In embodiments, measuring binding activity comprises measuring a signal from a secondary antibody that binds to an anti-SHANK3 antibody or an anti-Homer antibody (primary antibody) bound to the SHANK3 / Homer-GKAP complex formed on the assay substrate. Antibodies that bind other SHANK3 associated peptides such as SAPAP and GRIP may also be used.

[0126] In embodiments, measuring binding activity comprises measuring a signal from a secondary antibody that binds to an anti-SHANK3 antibody that detects the SHANK3 protein, including the mini-SHANK3 protein, bound to the GKAP peptide. In embodiments, the anti- SHANK3 antibody binds to miniSHANK3. In embodiments, the anti-SHANK3 antibody is Santa Cruz antibody SC-377470 (mouse monoclonal I G2a provided at 200 pg / mL) or an antibody that competes for binding to SHANK3 with the Santa Cruz antibody SC-377470 or other SHANK3 binding antibody. Appropriate anti-SHANK3 antibodies can be screened for binding to the SHANK3-GKAP complex in the assay system using methods known in the art. A secondary antibody, for example an anti-mouse secondary antibody, conjugated to a detectable label can be used in the assay.

[0127] In embodiments, measuring binding activity comprises measuring a signal from a secondary antibody that binds to the species of antibody that is the anti-Homer antibody.Because the SHANK3 that binds to the GKAP peptide associates with Homerl and other SHANK3 associated proteins, Homer 1 antibodies may be used to detect binding of SHANK3 to the GKAP peptide in the assay. A Homer antibody can be selected which binds and detects Homerl when associated with SHANK3 (including mini-SHANK3) that is bound to the GKAP peptide in the assay format. In embodiments, the anti-Homer antibody is Abeam ab97593 (rabbit polyclonal to Homerl) or an antibody that competes for binding to Homer with Abeam ab97593. In embodiments, the Abeam ab97593 antibody is used at dilutions greater than 1 / 100.

[0128] Other polyclonal and / or monoclonal antibodies to SHANK3 and / or Homer may be used in the disclosed methods and can be identified using methods for screening for antibody specific binding known in the art.

[0129] The binding of the anti-SHANK3 or anti-Homer antibody is detected by a secondary antibody which binds to the anti-SHANK3 or anti-Homer antibody. In embodiments, the binding activity is detected with anti-SHANK3 or anti-Homer antibodies that detect and bind to the SHANK3 (including mini-SHANK3)-Homer-GKAP complex formed in the assay and then, in embodiments, detectably labeled secondary antibodies that bind to the anti-SHANK3 or anti-Homer antibodies. Secondary antibodies are a population of immunoglobulins, including, for example, IgGs, generated in a host species that detect and bind to antibodies from a second species and can be detectably labeled. The secondary antibody detects antibodies of the species from which the anti-SHANK3 or anti-Homer antibody is derived. In embodiments, where theanti-SHANK3 or anti-Homer antibody is a rabbit antibody, the secondary antibody is a goat antirabbit antibody. In embodiments, where the anti-SHANK3 or anti-Homer antibody is a mouse antibody, the secondary antibody is a goat anti-mouse antibody.

[0130] In embodiments, the secondary antibody is Thermofisher, G21234 antibody (goat anti-rabbit HRP). In embodiments, the second antibody is ThermoFisher A-16035 antibody (donkey anti-rabbit IgG HRP). In embodiments, the second antibody is ThermoFisher A16072 (goat anti-mouse IgG HRP).

[0131] Detection Systems

[0132] Detection and quantitation of SHANK3 binding may be carried out by detection of a signaling molecule and quantitation of the signal from that molecule in the assay. In embodiments, the secondary antibody is conjugated to a signaling molecule that is detectable and quantifiable. In embodiments, the signaling molecule is a fluorophore. In embodiments, the signaling molecule is an enzyme. In embodiments, the enzyme is horseradish peroxidase (HRP). In embodiments, the enzyme is alkaline phosphatase (AP).

[0133] In embodiments, a signal is produced by interaction of the signaling molecule and a detector molecule. In embodiments, the detector molecule is 3,3-,5,5-Tetranmethylbenzidine (TMB). TMB is a chromogenic substrate that acts as a hydrogen donor for the reduction of hydrogen peroxide to water by horseradish peroxidase. The resulting product takes on a blue color, the absorbance of which can be read at 370 and 650 nm. The reaction can be halted by addition of acid or another stop reagent. Using sulfuric acid turns TMB yellow, with a peak absorbance of 450 nm. The amount of converted TMB may be indexed by the amount of 450 nm light it absorbs.

[0134] In embodiments, the detector molecule is a fluorophore. In embodiments, the detector molecule is QuantaBlu. QuantaBlu Fluorogenic Peroxidase Substrate Kit (Thermo Fisher Scientific) is a three-component kit containing proprietary fluorescent substrate, peroxide and stop solutions. QuantaBlu fluorescence is measured using a microplate reader set to an excitation of 325 nm and an emission of 420 nm and can be expressed as relative fluorescent units (RFUs). Other fluorescent signaling molecules are known in the art.

[0135] In embodiments, measuring binding activity of SHANK3 or SHANK3 associated proteins to the rGKAP peptide comprises measuring HRP activity with QuantaBlu.Potency Quantitation

[0136] To assess potency, the SHANK3 binding that is measured is compared against values obtained with serial dilutions of a known reference standard or interim reference standard. In embodiments, the reference standard is diluted to 3.0* 1012vg / mL with transduction media. The diluted vectors are then used to prepare five additional 1.5-fold serial dilutions, using transduction media as the diluent. The serial dilutions of the reference standard are assessed in the assay and the output is quantitated, including to generate a standard reference curve.

[0137] The relative activity of the sample is determined by first subtracting the average output, such as the relative fluorescence units (RFU), of the assay blank wells from the measured output of the samples and the reference standard wells. Using biostatistical software, such as SoftMax software or parallel-line analysis (PLA), the assay blank adjusted output (e.g., RFU) is plotted against the corresponding dilution to generate, for example, six-point dose response curves for the samples and the reference standard. A common slope is calculated for the reference standard and sample curves, which is used to constrain the fit to the resulting model for the reference standard and sample curves. In the constrained model, the difference in y-intercept between the sample curves and the standard curve is used to calculate the activity of the samples relative to that of the reference standard.Assay Qualification

[0138] In embodiments, the potency assay for quantitating mini-SHANK3 or SHANK3 binding activity is accurate, precise (repeatable), precise (intermediate), linear, has adequate range, is specific for mini-SHANK3 gene therapy and is able to detect changes in potency when AAV comprising mini-SHANK3 have been subjected to thermal stress. In embodiments, the method for quantitating miniSHANK3 binding activity have the features outlined in Table A and / or Table B.Table A - Qualification of Potency AssayTable B. Qualification of Potency Assay

[0139] In embodiments, the potency assay for quantitating mini-SHANK3 binding activity is accurate and has a % recovery from about 110% to about 115%, from about 111% to about 114%, or from about 112% to about 113%. In embodiments, the potency assay for quantitating mini-SHANK3 binding activity is accurate and has a % recovery of about 110%, about 111%, about 112%, about 113%, about 114% or about 115%.

[0140] In embodiments, the potency assay for quantitating mini-SHANK3 binding activity is precise (repeatable) with a percentage difference from identical samples of from about 5% to about 13%, from about 5% to about 12%, from about 5% to about 11%, from about 5% to about 10%, from about 5% to about 9% from about 5% to about 8%, or from about 5% to about 7%. In embodiments, the potency assay for quantitating mini-SHANK3 binding activity is accurate and precise (repeatable) with a percentage difference from identical samples of about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12% and about 13%.

[0141] In embodiments, the potency assay for quantitating mini-SHANK3 binding activity has an intermediate precision (%CV) of from about 1% to about 10%, about 2% to about 10%, about 3% to about 9%, about 4% to about 8%, or about 5% to about 7%. In embodiments, the potency assay for quantitating mini-SHANK3 binding activity has an intermediate precision (%CV) of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9% or about 10%.

[0142] In embodiments, the potency assay for quantitating mini-SHANK3 binding activity is linear and has an R2of about 0.98. In embodiments, the potency assay for quantitating mini-SHANK3 binding activity is linear and has an R2of about 0.99.

[0143] In embodiments, the potency assay for quantitating mini-SHANK3 binding activity has an adequate range of greater than equal to about 50% to less than or equal to 140%.

[0144] In embodiments, the potency assay for quantitating mini-SHANK3 binding activity is specific for mini-SHANK3 gene therapy and / or is able to detect changes in potency in AAV comprising mini-SHANK3 have been subjected to thermal stress. miniSHANK3 proteins

[0145] The SHANK family of proteins (e.g., SHANK1, SHANK2, and SHANK3) are master scaffolding proteins that tether and organize scaffolding proteins at the synapses of excitatory neurons. Members of this family share at least five main domain regions: N-terminal ankyrin repeats, SH3 domain, PDZ domain, proline-rich region, and a C-terminal SAM domain. Through these functional domains, SHANK proteins interact with many postsynaptic density (PSD) proteins. Without wishing to be bound by any theory, SHANK proteins can bind to SAPAP which in turn binds to PSD95 to form the PSD95 / SAPAP / SHANK postsynaptic complex. Together, these multidomain proteins are proposed to form a key scaffold, orchestrating the assembly of the macromolecular postsynaptic signaling complex at glutamatergic synapses. This complex has been shown to play important roles in targeting, anchoring, and dynamically regulating synaptic localization of neurotransmitter receptors and signaling molecules. In another example, the SHANK family of proteins is connected to the mGluR pathway through its binding to Homer.

[0146] In some embodiments, the present disclosure relates to SHANK proteins that are capable of restoring synaptic activity in subjects with disrupted SHANK protein activity. In some embodiments, the disrupted SHANK protein activity is present in subjects who have neurodevel opmental disorders, an autism spectrum disorder (ASD), and / or Phelan-McDermid syndrome. In some embodiments, the SHANK proteins associated with the present disclosure are SHANK1 proteins. In some embodiments, the present disclosure relates to expression in a subject in need thereof a polynucleotide encoding SHANK 1 or a variant of SHANK 1. In some embodiments, the SHANK proteins in the present disclosure are SHANK2 proteins. In some embodiments, the present disclosure relates to expression in a subject in need thereof a polynucleotide encoding SHANK2 or a variant of SHANK2. In some embodiments, the SHANK proteins in the present disclosure are SHANK3 proteins. In some embodiments, the present disclosure relates to expression in a subject in need thereof a polynucleotide encoding SHANK3or a variant of SHANK3. It should be appreciated that SHANK proteins associated with the present disclosure can include any SHANK protein, including variants or fragments thereof, that function as scaffolding proteins at the synapses of excitatory neurons.

[0147] Disclosed herein are polynucleotides encoding SHANK proteins (SHANK 1, SHANK 2, and SHANK3) and miniSHANK3 proteins for use in gene therapy.

[0148] The SHANK3 full length mouse protein sequence corresponding to GenBank Accession No. BAE16756.1 is provided by SEQ ID NO: 5.

[0149] In some embodiments, the SHANK3 full length mouse protein sequence corresponding to SEQ ID NO: 5 is encoded by a nucleic acid sequence corresponding to GenBank Accession No. NM_021423, provided by SEQ ID NO: 7.

[0150] The SHANK3 full length human protein sequence corresponding to GenBank Accession No. Q9BYB0.3 is provided by SEQ ID NO: 6.

[0151] In some embodiments, the SHANK3 full length human protein sequence corresponding to SEQ ID NO: 6 is encoded by a nucleic acid sequence corresponding to GenBank Accession No. NM_001372044, provided by SEQ ID NO: 8.

[0152] The full-length SHANK3 protein comprises multiple domains and is encoded by a gene that is about 5.2 Kb in size. Due to its size, it is difficult to deliver full-length SHANK3 to a tissue or cell of interest via an AAV vector. As reported in PCT Publication No.W02022 / 040239, entitled “SHANK3 Gene Therapy Approaches,” which is incorporated by reference herein in its entirety, specific domains can be removed or truncated from the full- length SHANK3 protein to produce MiniSHANK3 that is efficacious in restoring SHANK3 activity in excitatory neurons. SHANK proteins (e.g., SHANK3 proteins) encoded by polynucleotides described herein can be miniaturized to form a shortened variant of the native, full length SHANK3 protein. As disclosed herein, a miniaturized SHANK3 protein, or a DNA construct encoding the miniaturized Shank3 protein, are referred to interchangeably as “miniSHANK3” or “MiniSHANK3.” MiniSHANK3 proteins include shortened or mutated versions of SHANK3 that have at least some SHANK3 activity, for example, when MiniSHANK3 is introduced into neurons, it reduces the effects of SHANK3 mutations,

[0153] SHANK3 activity” includes, for example, the activity when introduced into, including by gene therapy, an organism, such as a mouse, non-human primate (NHP) or human, including the neurons of an organism, which is deficient for SHANK3 or had reduced SHANK3activity, that ameliorates the effects of that SHANK3 deficiency or reduction. The activity can be assessed in SHANK3 deletion cells or SHANK3 deficient animal models such as described in Examples 1-3 herein.

[0154] In some embodiments, the SHANK3 protein disclosed herein is expressed from a miniaturized SHANK3 DNA construct or expression cassette. In some embodiments, the variant SHANK3 DNA constructs and the SHANK3 proteins disclosed herein (MiniSHANK3) comprise fewer domains than the full-length SHANK3 gene and protein but have SHANK3 activity, including SHANK3 binding activity. In some embodiments, the miniSHANK3 protein disclosed herein is encoded by a non-naturally occurring polynucleotide.

[0155] miniSHANK3 proteins encoded by polynucleotides described herein can include one or more SHANK3 protein domains. For example, miniSHANK3 proteins and SHANK3 proteins include one or more of an SH3 domain, a PDZ domain, a Homer binding domain, a Cortactin domain, a SAM domain, and / or an ankyrin repeat domain. In embodiments, the miniSHANK3 protein is the amino acid sequence of SEQ ID NO: 9, 10, 11, or 12. In embodiments, the miniSHANK3 protein is the amino acid sequence of SEQ ID NO: 10 or 12. In embodiments, the miniSHANK3 protein is encoded by the sequence of SEQ ID NO: 1, 2, 3 or 4. In embodiments, the miniSHANK3 protein is encoded by the sequence of SEQ ID NO: 2 or 4.

[0156] SHANK3 proteins include the following domains: an ankyrin repeat domain, an SH3 domain, a PDZ domain, a proline rich domain, Homer binding domain, a Cortactin binding domain, and a SAM domain. In embodiments, the SHANK3 protein can be a mouse or a human SHANK3 protein. In embodiments, the SHANK3 has an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6. In embodiments, the SHANK3 is encoded by a nucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 8. In other embodiments, the SHANK3 protein is an isoform. In some embodiments, the isoform is SHANK3a, SHANK3b, SHANK3c, SHANK3d, SHANK3e, SHANK3f or any other isoform known in the art.

[0157] In some embodiments, the SH3 domain comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to residues 474-525 of SEQ ID NO: 6 or residues 473-524 of SEQ ID NO: 5. In some embodiments, the SH3 domain comprises at least 90% identity to residues 474-525 of SEQ ID NO: 6. In some embodiments, the SH3 domain comprises at least90% identity to residues 473-524 of SEQ ID NO: 5. In some embodiments, the SH3 domain comprises residues 474-525 of SEQ ID NO: 6. In some embodiments, the SH3 domain comprises residues 473-524 of SEQ ID NO: 5. In some embodiments, the SH3 domain can comprise any percent identity to residues 474-525 of SEQ ID NO: 6 suitable for construction of the MiniSHANK3. In some embodiments, the SH3 domain can comprise any percent identity to residues 473-524 of SEQ ID NO: 5 suitable for construction of the MiniSHANK3.

[0158] In some embodiments, the PDZ domain comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to residues 573-662 of SEQ ID NO: 6 or residues 572-661 of SEQ ID NO: 5. In some embodiments, the PDZ domain comprises at least 90% identity to residues 573-662 of SEQ ID NO: 6. In some embodiments, the PDZ domain comprises at least 90% identity to residues 572-661 of SEQ ID NO: 5. In some embodiments, the PDZ domain comprises residues 573-662 of SEQ ID NO: 6. In some embodiments, the PDZ domain comprises residues 572-661 of SEQ ID NO: 5. In some embodiments, the PDZ domain can comprise any percent identity to residues 573-662 of SEQ ID NO: 6 suitable for construction of the MiniSHANK3. In some embodiments, the PDZ domain can comprise any percent identity to residues 572-661 of SEQ ID NO: 5 suitable for construction of the MiniSHANK3.

[0159] In some embodiments, the Homer binding domain comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to residues 1294-1323 of SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, the Homer domain comprises at least 90% identity to residues 1294-1323 of SEQ ID NO: 5. In some embodiments, the Homer domain comprises at least 90% identity to residues 1294-1323 of SEQ ID NO: 6. In some embodiments, the Homer domain comprises residues 1294-1323 of SEQ ID NO: 5 or 6. In some embodiments, the Homer domain can comprise any percent identity to residues 1294-1323 of SEQ ID NO: 5 or SEQ ID NO: 6 suitable for construction of the MiniSHANK3.

[0160] In some embodiments, the Cortactin binding domain comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is100% identical, including all values in between, to residues 1400-1426 of SEQ ID NO: 5 or 6. In some embodiments, the Cortactin binding domain comprises at least 90% identity to residues 1400-1426 of SEQ ID NO: 5. In some embodiments, the Cortactin binding domain comprises at least 90% identity to residues 1400-1426 of SEQ ID NO: 6. In some embodiments, the Cortactin binding domain comprises residues 1400-1426 of SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, the Cortactin binding domain can comprise any percent identity to residues MOO- 1426 of SEQ ID NO: 5 or SEQ ID NO: 6 suitable for construction of the MiniSHANK3.

[0161] In some embodiments, the SAM domain comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to residues 1664-1729 of SEQ ID NO: 6 or to residues 1663- 1728 of SEQ ID NO:5. In some embodiments, the SAM binding domain comprises at least 90% identity to residues 1664-1729 of SEQ ID NO: 6. In some embodiments, the SAM binding domain comprises at least 90% identity to residues 1663-1728 of SEQ ID NO: 5. In some embodiments, the SAM domain comprises residues 1664-1729 of SEQ ID NO: 6. In some embodiments, the SAM domain comprises residues 1663-1728 of SEQ ID NO: 5. In some embodiments, the SAM binding domain can comprise any percent identity to residues 1664-1729 of SEQ ID NO: 6 suitable for construction of the MiniSHANK3. In some embodiments, the SAM binding domain can comprise any percent identity to residues 1663-1728 of SEQ ID NO: 5 suitable for construction of the MiniSHANK3.

[0162] In some embodiments, the ankyrin repeat domain comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to residues 148-345 of SEQ ID NO: 6 or to residues 147-313 of SEQ ID NO: 5. In some embodiments, the ankyrin repeat domain comprises at least 90% identity to residues 148-345 of SEQ ID NO: 6. In some embodiments, the ankyrin repeat domain comprises at least 90% identity to residues 147-313 of SEQ ID NO: 5. In some embodiments, the ankyrin repeat domain can comprise any percent identity to residues 148-345 of SEQ ID NO: 6 suitable for construction of the MiniSHANK3. In some embodiments, the ankyrin repeat domain can comprise any percent identity to residues 147-313 of SEQ ID NO: 5 suitable for construction of the MiniSHANK3.

[0163] In some embodiments, the MiniSHANK3 protein is less than 65% identical to SEQ ID NO: 5 over the full length of SEQ ID NO: 5. In some embodiments, the MiniSHANK3 protein is less than 65% identical to SEQ ID NO: 6 over the full length of SEQ ID NO: 6. As used herein, “less than 65%” includes any percent identity less than 65% that is suitable for construction of the MiniSHANK3. In some embodiments, the MiniSHANK3 protein is less than 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11% or 10% identical to SEQ ID NO: 5 over the full length of SEQ ID NO: 5. In some embodiments, the MiniSHANK3 protein is less than 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11% or 10% identical to SEQ ID NO: 6 over the full length of SEQ ID NO: 6. In some embodiments, the MiniSHANK3 protein is at least about 35%, at least about 40%, at least about 45%, or at least about 50% identical to SEQ ID NO: 5 or SEQ ID NO: 6 over the full length of SEQ ID NO: 5 or SEQ ID NO: 6.

[0164] In some embodiments, the MiniSHANK3 protein comprises an amino acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, including all values in between, to any one of SEQ ID NOs: 9-12, provided in Table 26.

[0165] In some embodiments, the MiniSHANK3 protein comprises or consists of any one of the amino acid sequences of SEQ ID NOs: 9-12. In some embodiments, SEQ ID NO: 9 is encoded by SEQ ID NO: 1. In some embodiments, SEQ ID NO: 10 is encoded by SEQ ID NO: 2. In some embodiments, SEQ ID NO: 11 is encoded by SEQ ID NO: 3. In some embodiments, SEQ ID NO: 12 is encoded by SEQ ID NO: 4.

[0166] In some embodiments, the MiniSHANK3 protein comprises an ankyrin repeat domain. In certain embodiments in which the MiniSHANK3 protein comprises an ankyrin repeat domain, the MiniSHANK3 protein comprises SEQ ID NO: 11 and / or SEQ ID NO: 12.

[0167] In other embodiments, the MiniSHANK3 protein does not comprise an ankyrin repeat domain. In certain embodiments in which the MiniSHANK3 protein does not comprise an ankyrin repeat domain, the MiniSHANK3 protein comprises SEQ ID NO: 9 and / or SEQ ID NO: 10.

[0168] In some embodiments, the sequences of polynucleotides encoding MiniSHANK3 proteins associated with the disclosure comprise at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or are 100% identical, including all values in between, to any one of SEQ ID NOs: 1-4, and encode one or more proteins with SHANK3 activity. In some embodiments, the sequences of polynucleotides encoding MiniSHANK3 proteins associated with the disclosure comprise at least 90% identity to any one of SEQ ID NOs: 1-4, and encode one or more proteins with SHANK3 activity. In some embodiments, the sequences of polynucleotides encoding MiniSHANK3 proteins associated with the disclosure comprise any one of SEQ ID NOs: 1-4. In some embodiments, any one of SEQ ID NOs: 1-4 encodes one or more proteins with SHANK3 activity.

[0169] In some embodiments, the MiniSHANK3 is encoded by any one of SEQ ID NOs: 1-4, provided in Table 26. SEQ ID NO: 1 and SEQ ID NO: 3 correspond to mouse MiniSHANK3 nucleic acid sequences, while SEQ ID NO: 2 and SEQ ID NO: 4 correspond to human MiniSHANK3 nucleic acid sequences. SEQ ID NO: 1 and SEQ ID NO: 2 encode MiniSHANK3 proteins that do not comprise an ankyrin repeat domain or the N-terminal domain. SEQ ID NO: 3 and SEQ ID NO: 4 encode MiniSHANK3 proteins that comprise an ankyrin repeat domain and the N-terminal domain.

[0170] Polynucleotides described herein that encode MiniSHANK3 proteins encode proteins that have SHANK3 activity.

[0171] As disclosed herein, “identity” of sequences refers to the measurement or calculation of the percent of identical matches between two or more sequences with gap alignments addressed by a mathematical model, algorithm, or computer program that is known to one of ordinary skill in the art. The percent identity of two sequences (e.g., nucleic acid or amino acid sequences) may, for example, be determined using Basic Local Alignment Search Tool (BLAST®) such as NBLAST® and XBLAST® programs (version 2.0). Alignment technique such as Clustal Omega may be used for multiple sequence alignments. Other algorithms or alignmentmethods may include but are not limited to the Smith-Waterman algorithm, the Needleman- Wunsch algorithm, or Fast Optimal Global Sequence Alignment Algorithm (FOGSAA).

[0172] In some embodiments, a polynucleotide encoding the SHANK protein as disclosed herein (SHANK 1, SHANK2, SHANK3) is less than about 4.6 kb, about 4.5 kb, about 4.4 kb, about 4.3 kb, about 4.2 kb, about 4.1 kb, about 4.0 kb, about 3.9 kb, about 3.8 kb, about3.7 kb, about 3.6 kb, about 3.5 kb, about 3.4 kb, about 3.3 kb, about 3.2 kb, about 3.1 kb, about3.0 kb, about 2.9 kb, about 2.8 kb, about 2.7 kb, about 2.6 kb, about 2.5 kb, about 2.4 kb, about2.3 kb, about 2.2 kb, or about 2.1 kb in size. In some embodiments, the polynucleotide encoding the SHANK protein as disclosed herein (SHANK 1, SHANK2, SHANK3) can be in any size that is suitable for the methods and vectors disclosed in the present disclosure. In particular embodiments the SHANK polynucleotide encodes SHANK3.Viral Vectors

[0173] In some embodiments, the miniSHANK3 transgene coding sequence in the recombinant AAV vector is operably linked to regulatory sequences for tissue-specific gene expression. In some cases, the tissue-specific regulatory sequences bind tissue-specific transcription factors that induce transcription in a tissue specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art. In some embodiments, the tissue-specific regulatory sequence can be a Syn promoter (e.g., hSynl). In some embodiments, the hSynl promoter comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to the sequence of SEQ ID NO: 14. In some embodiments, the hSynl promoter comprises the nucleic acid sequence of SEQ ID NO: 14. In some embodiments, the tissue-specific regulatory sequence can be any promoter or enhancer that is neuron-specific and is suitable for the treatments described herein.

[0174] In some embodiments, a miniSHANK3 transgene encoding a nucleotide sequence comprising SEQ ID NO: 2 or SEQ ID NO: 4 in a recombinant AAV vector is operably linked to a promoter, including the hSyn promoter, and is flanked by AAV ITRs. In some embodiments, a miniSHANK3 transgene encoding a nucleotide sequence comprising SEQ ID NO: 1 or SEQ ID NO: 3 in a recombinant AAV vector is operably linked to a promoter and is flanked by AAV ITRs.

[0175] In some embodiments, a miniSHANK3 transgene comprising a nucleotide sequence encoding an amino acid sequence comprising SEQ ID NO: 10 or SEQ ID NO: 12 in a recombinant AAV vector is operably linked to a promoter, including the hSyn promoter, and is flanked by AAV ITRs. In some embodiments, a miniSHANK3 transgene comprising a nucleotide sequence encoding an amino acid sequence comprising SEQ ID NO: 9 or SEQ ID NO: 11 in a recombinant AAV vector is operably linked to a promoter and is flanked by AAV ITRs.

[0176] Aspects of the disclosure relate to assays of potency of recombinant AAV vectors expressing miniSHANK3 transgenes. In some embodiments, a miniSHANK3 transgene is flanked by AAV ITRs. In some embodiments, the AAV ITRs comprise AAV2 ITRs. In some embodiments, the AAV ITRs comprise AAV1 ITRs. In some embodiments, the AAV ITRs comprise AAV5 ITRs. In some embodiments, the AAV ITRs comprise AAV6 ITRs. In some embodiments, the AAV ITRs comprise AAV8 ITRs. In some embodiments, the AAV ITRs comprise AAV9 ITRs. In some embodiments, the AAV ITRs comprise rhlO ITRs. In some embodiments, the AAV ITRs may include one or more modified ITRs which generate self- complementary AAV genomes.

[0177] In some embodiments, the recombinant AAV vector comprises a 5’ AAV2 ITR and a 3’ AAV2 ITR. In some embodiments, the 5’ AAV2 ITR comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to the sequence of SEQ ID NO:18. In some embodiments, the 3’ AAV2 ITR comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is100% identical, including all values in between, to the sequence of SEQ ID NO: 19. In some embodiments, the 5’ AAV2 ITR comprises the nucleic acid sequence of SEQ ID NO: 18. In some embodiments, the 3’ AAV2 ITR comprises the nucleic acid sequence of SEQ ID NO: 19.

[0178] It should be appreciated that AAV vectors described herein can include DNA constructs or expression cassettes that comprise a transgene such as MiniSHANK3, 5’ and / or 3’ ITRs, promoters, introns, and / or other associated regulatory elements that are known in the art.

[0179] In some embodiments, the AAV vector comprises a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), which may enhance miniSHANK3 transgene expression. In some embodiments, the WPRE comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to the sequence of SEQ ID NO: 15. In some embodiments, the WPRE comprises the nucleic acid sequence of SEQ ID NO: 15.

[0180] In some embodiments, the AAV vector comprises an untranslated portion such as an intron or a 5’ or 3’ untranslated region. In some embodiments, the intron may be located between the promoter / enhancer sequence and the miniSHANK3 transgene.

[0181] In some embodiments, the AAV vector used herein may be a self-complementary vector.

[0182] SEQ ID NO: 13 comprises a human MiniSHANK3 gene, a 5’-ITR, a 3’-ITR, a WPRE, an hGH poly A, and a hSynl promoter. SEQ ID NO: 21 comprises a human MiniSHANK3 gene, a 5’-ITR, a 3’-ITR, a WPRE, an hGH poly A, and a hSynl promoter.

[0183] In some embodiments, the inverted terminal repeat (ITR) sequences comprise about 145 nucleotides each. These elements may be useful in cis for effective replication and encapsidation. A skilled person in the art would appreciate that any elements of AAV vectors known in the art may be compatible with aspects of the disclosure. One of skill in the art would also appreciate that any of the polynucleotide sequences described herein that encode a functional MiniSHANK3 protein can be expressed in a DNA construct or expression cassette for AAV delivery. These DNA constructs or expression cassettes may include one or more of the elements described herein. For example, in some embodiments a coding sequence comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to any one of SEQ ID NOs: 1-4 is expressed in a DNA construct or expression cassette. In some embodiments a coding sequence comprising the sequence of any one of SEQ ID NOs: 1-4 is expressed in a DNA construct or expression cassette. In some embodiments, the DNA construct or expression cassette includes one or more elements such as a promoter, a 5’-ITR, a 3’-ITR, a Synl promoter, a WPRE, an hGH polyA. Cis plasmids for production of the recombinant AAV virions may have elements such as origin of replications and antibiotic resistance markers, for example, an Fl origin, a NeR / KanR marker and / or a PUC origin.

[0184] Expression cassettes described herein may comprise a polyA signal. In some embodiments, the polyA signal is the hGH polyA signal. In some embodiments, the polyA signal comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to the sequence of SEQ ID NO: 16. In some embodiments, the polyA signal comprises the nucleic acid sequence of SEQ ID NO: 16.

[0185] In some embodiments, a recombinant AAV vector associated with the disclosure includes a nucleic acid sequence encoding a MiniSHANK3 protein operably linked to regulatory elements that promote CNS expression and flanking ITRs that comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, including all values in between, to the sequence of SEQ ID NO: 13 or SEQ ID NO: 21, provided in Table 26. In some embodiments, a recombinant AAV vector comprises a sequence corresponding to SEQ ID NO: 13 or SEQ ID NO: 21, which encode a MiniSHANK3 protein comprising the sequence of SEQ ID NO: 10 and comprises regulatory elements, including a hSynl promoter, an WPRE element and a polyA signal sequence and flanking ITR sequences. In some embodiments, a recombinant AAV vector comprising the sequence of SEQ ID NO: 13 or SEQ ID NO: 21 may be delivered to a human subject in need thereof and may be suitable for treating a human subject who has a neurodevel opmental disorder.

[0186] As one of ordinary skill in the art would appreciate, any method known in the art for designing AAV vectors for clinical use, and for delivery of AAV vectors, may be compatible with aspects of the disclosure. For example, non-limiting examples of disclosure related to AAV vectors and delivery are provided in and incorporated by reference from U.S. Patent No.7,906,111, entitled “Adeno-associated virus (AAV) clades, sequences, vectors containing same, and uses therefor” and U.S. Patent No. 9,834,788, entitled “AAV -vectors for use in gene therapy of choroideremia,” each of which is incorporated by reference herein in its entirety.

[0187] In some embodiments, the recombinant AAV vector encoding a MiniSHANK3 protein for AAV delivery encodes a protein with a sequence that comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or is 100% identical, to any one of SEQ ID NOs: 9-12, provided in Table 26.

[0188] The present disclosure provides recombinant AAV virions comprising: (1) recombinant AAV vectors described herein and (2) an AAV9 capsid. In some embodiments, the AAV9 capsid comprises an amino acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical, to SEQ ID NO: 20. As used herein, a “virion” refers to a viral particle that includes genetic material (e g., RNA or DNA) and a capsid.

[0189] In some embodiments, an expression cassette disclosed herein can comprise SEQ ID NO: 17 or SEQ ID NO: 13 (with flanking ITR sequences) or SEQ ID NO: 21 (with flanking ITR sequences) as shown in Table 26.

[0190] In some embodiments, a recombinant vector comprising an expression cassette that comprises a polynucleotide encoding the SHANK3 protein (i.e., the miniSHANK3 DNA construct) can be expressed in a specific tissue or cell of interest. In some embodiments, the expression cassette or vector disclosed herein comprises a promoter. In some embodiments, the expression cassette or vector comprises a cell-type specific promoter. In some embodiments, the promoter is a human promotor. In some embodiments, the human promoter is human Synapsin 1 (hSynl). In some embodiments, the hSynl promotor has a polynucleotide sequence corresponding to SEQ ID NO: 14. In some embodiments, the human promoter can be any promotor that is known in the art and is suitable for expression of miniSHANK3. In some embodiments, the human promoter can be any promoter that has high specificity for neuronal tissues and cells. In some embodiments, the promoter can be a constitutive promoter. For example, the constitutive promoter can be a CAG promoter. As one of ordinary skill in the art would appreciate, any promoter may be used so long as the selected promoter is compatible with aspects of the disclosure.

[0191] The present disclosure provides methods of producing an AAV virion. In some embodiments, the method comprises culturing a host cell comprising an AAV vector described herein, an AAV cap (capsid protein) and an AAV9 rep (replication protein), and optionally one or more additional adenoviral helper functions, under conditions sufficient to produce the AAV virion; and isolating the AAV virion produced by the host cell. In some embodiments, the AAVcap encodes VP1, VP2, and / or VP3. In some embodiments, the rep encodes rep78, rep68, rep52, and / or rep40.EXAMPLES

[0192] In order that the invention described herein may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the systems and methods provided herein and are not to be construed in any way as limiting their scope.Example 1: Generation and Characterization of SHANK3 Knockout Cells[00193J U-87 MG cells (ATCC, HTB-14) a glioblastoma / astrocytoma cell line of human origin were selected for use as the parental cell for generation of SKO cells. These cells are permissive to AAV9 transduction, drive expression of proteins from the synapsin promoter, and express the SHANK3 binding partner, Homer. U-87 MG cells were used for generation and isolation of the SKO cells.

[0194] To determine the levels of recombinant miniSHANK3 protein expression, SKO cells and U-87MG cells were transfected with a plasmid encoding miniSHANK3. Duplicate cultures of SKO and U-87 MG cells were mock transfected (vehicle only) to serve as negative controls. After three days post-transfection, cells were lysed in either RIPA Buffer (Thermofisher, 89900) or NP40 Cell Lysis Buffer (Thermofisher, FNN0021) for immunoblotting to evaluate SHANK3 mini gene expression using SHANK3 -specific antibody (FIG. 1). SHANK3 was detected in Western blots as multiple isoforms with the largest being greater than 200 kDa. Lanes 1 and 2 contain RIPA generated lysates from mock transfected U-87MG and SKO cells, respectively. Comparison of the lysates from the two cell lines shows little difference for most of the bands; however, the intensity of the largest band is greater in the U-87MG lysates than the SKO lysates. Lanes 3 and 4 contain RIPA generated lysates of miniSHANK3 GOI plasmid transfected SKO and U-87MG cells, respectively. Lysates in Lanes 3 and 4 show the presence of the same antibody reactive bands as those observed in Lanes 1 and 2 with the addition of an intense band migrating at approximately 90 kDa. The 90 kDa band is the expected size of the SHANK3 minigene encoded by the miniSHANK3 GOI. Lanes 5 and 6 contain NP40 Cell Lysis Buffer generated lysates of miniSHANK3 GOI plasmid transfected SKO and U-87MG cells, respectively. Lysates in Lanes 5 and 6 show the presence of the same antibody reactive bands atsimilar intensities as observed in Lanes 3 and 4 with the exception of the band migrating at approximately 200 kDa, which is of higher intensity in Lanes 5 and 6. Because lysates from mock transfected cells lysed with NP40 Cell Lysis Buffer were not analyzed it is unclear if the increased intensity of the 200 kDa band is due to transfection or the use of NEMO Cell Lysis Buffer.

[0195] SKO cells showed distinct morphology compared to the parental U-87 MG cells. To determine if the change in morphology was in response to loss of SHANK3, the SKO cells were transduced with miniSHANK3 or vehicle control. Microscopic images of the cells were taken 4 days post-transduction. Untransduced SKO cells formed numerous masses of cells after 4 days of culture. By comparison, SKO cells transduced with miniSHANK3 were observed primarily as isolated cells with very few masses of cells present. (FIG. 2) The morphology of miniSHANK3 transduced cells is similar to that of U-87MG cells (data not shown).

[0196] Based on the Western blot results it is unclear if the level of SHANK3 is reduced in the SKO cells relative to the U-87MG cells; however, the protein expressed from the SHANK3 minigene is detected in miniSHANK3 transfected cells by Western blotting with SHANK3 -specific antibody. Additionally, this experiment demonstrated that RIPA and NP40 containing buffers are suitable for solubilizing SHANK3. Lastly, SKO cells differ in phenotype from the U-87MG from which they originate and the U-87MG like morphology can be restored in SKO cells by transfection of the SHANK3 minigene.Example 2: Comparing Human GKAP Peptide and Rat GKAP Peptide Capture of SHANK3

[0197] GKAP has been established as a SHANK3 interactor. To determine which GKAP sequence shows higher binding affinity to SHANK3 protein, GKAP binding assays were performed with GKAP peptides conjugated to biotin at the N-terminus by an SS linker. Peptide sequences and their reconstitution conditions are listed in Table 2.Table 2. GKAP Peptide Sequences and Reconstitution Conditions[00198J Assay conditions are listed in Table 3. Standard capacity, clear, polystyrene 96- well neutravi din-coated microplates were incubated with reconstituted peptides. In order to coat microplates, wells were incubated in lOOuL of 0. lug / rnL of either hGKAP (SEQ ID NO: 27) or rGKAP (SEQ ID NO: 24) peptides in protein free blocking buffer (PFBB) for 1 hour at room temperature. Following microplate incubation with the peptides, unbound peptides were removed by washing. Diluent or lysates from mock transfected and miniSHANK3 transfected SKO cells were incubated in the microplate for Ih, followed by incubation with SHANK3 -specific primary antibodies. Then, the microplate was washed and HRP-conjugated, secondary antibody was added to the plate. Following incubation of secondary antibody, unbound antibody was removed by washing and TMB substrate was added to the microplate. Stop solution was added to the microplate once sufficient color development occurred. The resulting absorbance was measured at 450 nm with background subtraction measurement using a wavelength of 650 nm (FIG. 3).Table 3. Assay Conditions

[0199] Usage of the hGKAP peptide in the assay resulted in higher SHANK3 -specific absorbance compared to mock transfected lysates and diluent only. Similarly, rGKAP peptide resulted in SHANK3 -specific absorbance from the mock transfected lysates and the diluent only. In addition, rGKAP peptides showed higher SHANK3 -specific absorbance compared to hGKAP. Lastly, there was little difference in the absorbance between the results using microplates coated with 0.1 pg / mL and 1 pg / mL of GKAP peptide. Thus, GKAP peptides, in particular rGKAP (SEQ ID NO: 24), are suitable for capture of mini SHANK3 in a microplate format.Example 3: Screening of GKAP-SHANK3 Binding Conditions

[0200] To determine optimal GKAP-SHANK3 binding conditions, GKAP binding assay (from example 2) was performed. Cell lysis buffer, peptide coating concentration and peptide coating time were evaluated.

[0201] Mock and miniSHANK3 transfected cells were lysed with either 0.5 mL Cell Lysis Buffer II (NP40 Cell Lysis Buffer) or RIPA Lysis Buffer (ThermoFisher #89900) per le5 cells. Lysate diluted 1 / 10 in PFBB prior to microplate loading.

[0202] Peptide coating concentration was optimized by coating each well with 100 pL of the following concentrations: 0.1 pg / mL, 0.01 pg / mL or 0.001 pg / mL of rat (SEQ ID NO: 24) or human GKAP peptide (SEQ ID NO: 27) in PFBB for 1 hour. Additionally, duplicate 0.1 pg / mL peptide wells were incubated for 2 hours at room temperature. Following incubation, microplates were washed and binding was detected as described previously. Assay conditions are detailed in Table 4.Table 4. Assay Conditions

[0203] Absorbance and signal to noise ratio (transfected: mock transfected) show that rGKAP peptide performs better than hGKAP peptide in the SHANK3 binding assay. Secondly, results in this experiment indicate that the peptide coating concentration should be > 0.1 pg / mL and that the use of CLBII is preferable to use of RIP A buffer for preparation of cell lysates. Lastly, these results indicate that a peptide coating time of > 1 hour or < 2 hours is satisfactory (FIG. 4)

[0204] Further optimization of the SHANK3 binding assay was completed by testing GKAP peptide concentration for plate coating, lysate incubation time, and antibody conditions. Plates were incubated 100 pL of either 0.1 pg / mL or 1.0 pg / mL of rGKAP (SEQ ID NO: 24) in PFBB for 1 hour at room temperature. 100 pL of lysate were incubated for 1, 2, or 3 hours at 37 °C. Lastly, binding efficacy of a cocktail of 2 anti-SHANK3 antibodies Santa Cruz SC-377470and #377088) was compared to single anti-SHANK3 antibody (Santa Cruz #SC-377470) Assay conditions are described in Table 5.Table 5. Assay Conditions

[0205] Results indicate that peptide concentration of 1.0 pg / mL for microplate coating is slightly better than 0.1 pg / mL. Furthermore, incubation of lysates for 1-2 hours results in higher signal to noise ratios than an incubation of 3 hours. Lastly, Santa Cruz antibody SC-377470 alone results in satisfactory detection of mini SHANK3 in the binding assay (FIG. 5).Example 4: Screening Antibodies for Detection of SHANK3 Binding Partners

[0206] GKAP binding assay was performed to identify an antibody that detects one of the proteins reported to interact with SHANK3.

[0207] Neutravi din-coated microplates were incubated with 100 pL / well of 1.0 pg / mL rGKAP (SEQ ID NO: 24) in PFBB for 1 hour at room temperature, and subsequently washed. Lysate was generated using 0.5mL of CLB II per 100,000 cells. Plates were then incubated with 100 pL lysate / well for 1.5 hours at 37C with mixing at 300 RPM. In this assay, antibodies specific for the SHANK3 binding partners mGluR5, Homer, and Sharpin were evaluated. In addition, the resulting absorbance from the anti-mouse and anti-rabbit secondary antibodies in the absence of detection antibodies was also determined. Assay conditions are described in Table 6.Table 6. Assay ConditionsTable 6.1 SHANK3 Binding Partner Screen Antibodies

[0208] Results indicate that the antibodies against mGluR5 and Sharpin would not be suitable for detecting a SHANK3 binding partner. By comparison, samples probed with Homerspecific antibodies show that wells incubated with transfected lysates produced a higher absorbance than wells incubated with mock transfected lysates. These data suggest that Homer binding to SHANK3 may be detected in this assay. Lastly, comparison of the secondary antibodies indicates that the anti-rabbit secondary results in more background absorbance than the anti-mouse secondary (FIG. 6).

[0209] Subsequently, other antibodies reactive to the SHANK3 binding partners mGluR5, Homer, and Sharpin were evaluated to determine optimal detection. Also, optimization of microplate type (high capacity binding vs standard capacity binding) and temperature (37°C vs room temperature) were also evaluated. Assay conditions are described in Table 7.Table 7. Assay Conditions

[0210] Antibodies against Homer were the most promising for detection of a SHANK3 binding partner. High capacity binding neutravidin and standard capacity binding neutravidin coated microplates performed similarly in the SHANK3 binding assay. The SHANK3 binding assay showed improved performance when incubations were performed at ambient temperature (FIG. 7).Example 5: Screening of Homer Reactive Antibodies

[0211] To identify optimal Homer antibodies for usage within the SHANK3 assay, several Homer antibodies were tested. The SHANK3 binding assay was performed as previously described; three Homer antibodies were evaluated (Abeam ab 184955, ThermoFisher H00009456 M13, Santa Cruz sc-17842) and absorbance was compared to SHANK3 detecting antibodies. Detailed assay conditions are displayed in Table 8.Table 8. Assay Conditions

[0212] The Homer antibody, abl84955 performs better than sc-17842 and H00009456 - M13 and produces the highest absorbance in transfected cells relative to mock transfected cells (FIG. 8). The secondary antibody, #G-21234 performs better than A-16035 for detection of Homer. As such, ab-97593 will be used for detection of SHANK3-bound Homer.Example 6: Optimizing Lysate Dilution in SHANK3 Bindins Assay

[0213] To determine the optimal cell lysate to protein free blocking buffer (PFBB) ratio for the SHANK3 binding assay, mock and transfected cell lysates were diluted in various concentrations (1 :2, 1 :4, 1 :8) prior to microplate incubation. Wells were either probed with SHANK3 or Homer antibodies. Homer primary antibodies were probed with either anti-rabbit IgG HRP diluted 1 / 2000 (ThermoFisher, G-21234 or A-16035). Assay was completed as described in Table 9.Table 9. Assay Conditions

[0214] Increased concentration of lysate increased SHANK3 and Homer detection in transfected cells relative to mock transfected cells (FIG. 9). Comparison of the secondary antibodies, used in conjunction with the anti-Homer antibodies, shows a lower absorbance for the Thermofisher, A16035 antibody (#1) than the Thermofisher, G21234 antibody (#2).Example 7: Optimizing MiniSHANK3 Transduction

[0215] In order to identify optimal cell density, transduction duration, and FBS concentration, SHANK3 knock out cells were transduced with 3 different doses of miniSHANK3 interim reference standard using various conditions to identify the optimum transduction conditions. Cells were seeded at a density of 5e3 or le4 cells per well. Transduction media contained 5% FBS or 10% FBS. Transductions were incubated for 3 days or 4 days at 37 °C with 5% CO2. Cell lysates were analyzed using the GKAP binding assay in conjunction with the anti-SHANK3 detection antibody.

[0216] Assay conditions are described in Table 10. Briefly, cells seeded at a density of le4 cells per well and transduced with MEM + 10% FBS had the highest absorbances for 3-day and 4-day transductions Furthermore, the results indicate that as FBS concentration decreased, the resulting absorbance also decreased. Cells transduced for 4 days resulted in higher absorbances for cells that were transduced using MEM + 10% media. There was little difference between 3- and 4-day transductions in the other experimental groups (FIGs. 10A-B).Table 10. Assay Conditions

[0217] Additional experiments were conducted to further optimize miniSHANK3 transduction and evaluate QuantaBlu fluorescent substrate to improve performance. Assay conditions are described in Table 11.Table 11. Assay Conditions

[0218] Briefly, SHANK3 knock out cells were transduced with 9 concentrations (2 -fold dilutions from 4el 1 - 1.56e9 vg per well) of miniSHANK3 interim reference standard. Cells were seeded at a density of le4, 3e4, 5e4, and 7e4 cells per well. The resulting cell lysates were analyzed using the GKAP binding assay in conjunction with the anti-SHANK3 detection antibody. Following washes to remove unbound secondary antibody, 0.100 mL per well of QuantaBlu substrate was incubated for 30 minutes at room temperature. The reaction was stopped by addition of 0.100 mL per well of QuantaBlu stop solution. Fluorescence was measured using a Molecular Devices iD5 microplate reader set to an excitation of 325 nm and an emission of 420 nm.

[0219] Cell densities from le4 - 5e4 cells per well result in the highest fluorescence and provide satisfactory dose response curves from 4el 1 to approximately lelO vg per well (FIG. 11).Example 8: Optimizing Detection Antibody Dilution

[0220] Previous results in examples demonstrate that that the anti-Homer antibody, ab97593 (Abeam) is the most suitable for detection of SHANK3-bound Homer in the GKAP binding assay. In the previous experiments, ab97593 was used at dilutions of 1 / 50 - 1 / 100. To determine if larger dilutions of antibody resulted in higher Homer-specific absorbance compared to background absorbance, ab97593 was evaluated at dilutions of 1 / 400, 1 / 800 and 1 / 1600. The GKAP binding assay conditions are described in Table 12.Table 12. Assay Conditions

[0221] The absorbance using anti-SHANK3 is consistent with previous results. Comparison of the results obtained with ab97593 indicates that the highest Homer-specific absorbance to background ratio was obtained with an antibody dilution of 1 / 1600 (FIG. 12). Increasing the dilution of ab97593 to greater than 1 / 100 resulted in higher Homer-specific absorbances compared to background.Example 9: Homer Bindins with a SHANK3 Bindins Mutant

[0222] Data presented herein demonstrate the detection of Homer in the GKAP binding assay. Because the level of Homer detected in the assay is higher in lysates produced from miniSHANK3 transfected cells it can be inferred that the detection of Homer is the result of its binding to miniSHANK3. However, it is possible that the increased level of Homer following transfection of SHANK3 is due to another mechanism. To demonstrate a direct interaction between Homer and miniSHANK3 in the GKAP binding assay, a miniSHANK3 protein containing two mutations (P131 IL and F134C) in the Homer binding domain was tested in the GKAP binding assay. These mutations were previously shown by Tu et al., 1999 to abrogate binding of Homer with SHANK3.

[0223] Lysates were generated from untransfected SHANK3 KO cells, SHANK3 KO cells transfected with a plasmid encoding wild type miniSHANK3 or SHANK3 KO cells transfected with a plasmid encoding miniSHANK3 containing the Homer binding mutations (HBM). Equal amounts of each lysate were tested in duplicate GKAP binding assays in which the detection antibody was either anti -Homer or anti-SHANK3. Additionally, fluorescence HRP substrate QuantaBlu was used in lieu of TMB substrate. Use of fluorescent substrates in ELISAs can provide higher signal to background levels than the measurement of absorbance at 450 nm in conjunction with TMB (Table 13).Table 13. Assay Conditions

[0224] Levels of Homer-specific fluorescence detected in mini SHANK3 -transfected lysates and miniSHANK3 HBM-transfected lysates indicate that detection of Homer in the GKAP binding assay occurs through the binding of miniSHANK3 to GKAP and the concomitant binding of Homer to miniSHANK3. In addition, use of a fluorescent HRP substrate in the GKAP binding assay provides improved signal to background ratios as compared to a previously used TMB -based substrate (FIG. 13).Example 10: Using Hydroxyurea to Increase Assay Sensitivity

[0225] In an effort to increase assay sensitivity, inclusion of hydroxyurea in the transduction media at final concentrations of 4 mM and 40 mM were evaluated.

[0226] SHANK3 knock out cells were transduced with 7 concentrations (1.5-fold dilutions from 2el 1 - 1.8el0 vg per well) of miniSHANK3 interim reference standard in the presence or absence of hydroxyurea. Following overnight incubation of the cells with transduction media (± hydroxyurea), transduction media was replaced with MEM + 10% FBS + 1% antibiotics. Cells were lysed 3 days after the media change and the resulting cell lysates were analyzed using the GKAP binding assay in conjunction with the anti-Homer detection antibody. Assay conditions are completely described in Table 14.Table 14. Assay Conditions

[0227] Transduced cells incubated with 4 mM hydroxyurea had increased miniSHANK3- associated Homer compared to cells incubated with transduction media without hydroxyurea. Furthermore, transduced cells incubated with 40 mM hydroxyurea had increased miniSHANK3- associated Homer compared to cells incubated with 4 mM hydroxyurea (FIG. 14).Example 11: Functional Activity by U-87 SHANK3 KO Transduction and ELISA

[0228] SHANK3 encodes a key postsynaptic scaffolding protein that is expressed in neuronal cells. As a ‘master’ scaffolding protein, it forms a key structural component of the postsynaptic density (PSD) of glutamatergic synapses, the main excitatory synapses in the brain (Drapeau, 2018). miniSHANK3 encodes for a miniaturized version of the SHANK3 gene that retains key binding domains (SH3, PDZ, Homer binding site, cortactin binding site, SAM domain) required for the proper anchoring of glutamate receptors to the PSD (FIG. 15).

[0229] A functional activity assay has been developed to demonstrate potency of miniSHANK3 via the SHANK3-Homerl binding mechanism of action. In vivo, the SHANK3- Homerl protein interaction is responsible for the stabilization of mGluR to the PSD (Tu, 1999), which supports the use of measuring miniSHANK3-Homerl binding as a functional activity assay for miniSHANK3. The miniSHANK3 functional activity assay for miniSHANK3 is measured by an ELISA specific for the SHANK3 binding partner, Homer 1, using miniSHANK3 transduced cells.

[0230] In this assay, cells of the U-87 MG cell line containing a deletion of the SHANK3 gene are seeded into 96-well, cell culture-treated microplates on the day of transduction. Six concentrations of miniSHANK3 test sample(s) and miniSHANK3 reference standard are prepared in cell culture medium containing hydroxyurea. Test sample dilutions, referencestandard dilutions, or cell culture medium (for preparation of assay blanks) are added to the cells and the microplate is incubated for approximately 96 hours. Ninety-six (96) hours posttransduction, the cells are lysed in buffer containing detergent and protease inhibitor. After lysis, lysates are added to 96-well microplates coated with peptide encoding the SHANK3 binding domain of guanylate kinase-associated protein (GKAP). Following incubation of the lysates in the GKAP-coated microplates and washes to remove unbound protein complexes, Homerl specific antibody is added to the microplate. After incubation of the Homerl specific antibody and washes to remove unbound antibody, horseradish peroxidase (HRP) conjugated secondary antibody is added to the plate. After incubation of the HRP-conjugated secondary antibody and washes to remove the unbound antibody, an HRP-specific substrate resulting in fluorescence is added to the microplate. The resulting fluorescence is measured, in RFU, using a microplate reader set to an excitation of 325 nm and an emission of 420 nm. The relative activity of the sample is determined by first subtracting the average RFU of the assay blank wells from the RFU of the samples and the reference standard wells. Using biostatistical software, the assay blank adjusted RFU is plotted against the corresponding dilution to generate six-point dose response curves for the samples and the reference standard. A common slope is calculated for the reference standard and sample curves, which is used to constrain the fit to the resulting model for the reference standard and sample curves. In the constrained model, the difference in y-intercept between the sample curves and the standard curve is used to calculate the activity of the samples relative to that of the reference standard.Table 15. Reagents for Functional Activity in AAV miniSHANK3 Transduced CellsTable 16. Buffers and Solutions for Functional Activity in AAV miniSHank3 Transduced CellsTable 17. Settings for Measuring Fluorescence for the AAV-miniSHANK3 Functional Activity in AAV-MiniSHANK3 transduced cells ELISA method

[0231] Sample and Standard Preparation

[0232] Reference standard and samples are diluted to 3. Ox 1012vg / mL with transduction media. The diluted vectors are then used to prepare 5 additional 1.5-fold serial dilutions, using transduction media as the diluent.Procedure

[0233] SHANK3 KO cells are passaged using aseptic technique. Cells are cultured in vented tissue culture-treated flasks with 10% FBS in MEM (pre- warmed to 37°C). For use in this assay, the cells are never expanded past 10 passages or cultured to a density > 3x 104viable cells per cm2. Trypsin EDTA at room temperature is used to detach the cell monolayer from the flask(s) and a final cell suspension at 4x 105viable cells / mL (10% FBS in MEM) is used to seed 100 pL of cells per well of a sterile, 96-well cell culture plate. Cells are only added to the wells being used for the assay and the outer perimeter of wells (column 1 and 12, row A and H) are excluded from use (200 pL PBS is added to the outer perimeter wells). All prepared dilutions (6 total) of samples and standards are plated in triplicate with 100 pL added per well containing cells. The non-transduced control is prepared by adding 100 pL of Transduction Media to three wells containing cells without vector.

[0234] The prepared cell culture plate is incubated on a microplate shaker for 60 minutes (300 rpm, 25°C or ambient temperature), followed by incubation in a humidified incubator (37°C, 5% CO2) for 16-24 hours. After incubation, the media is removed from the wells and replaced with 10% FBS in MEM (pre-warmed to 37°C). The cell culture plate is returned to a humidified incubator (37°C, 5% CO2) and incubated for an additional 66-78 hours. On the day of ELISA, after the final cell incubation, the media is removed from the wells and replaced with Lysis Buffer + Protease Inhibitor (for the wells containing cells). The cell culture plate is incubated on a microplate shaker for 30 minutes (300 rpm, 25°C or ambient temperature).

[0235] While the plate is incubating, the GKAP peptide-coated microplate (prepared using Neutravidin-coated microplate, Thermofisher / 15510, and GKAP Peptide Working Solution) is washed with TBS-T using a microplate washer. Protein-Free T20 (TBS) Blocking Buffer and cell lysate from the cell culture plate are added to each GKAP-coated well, with well locations of the standards, samples, and controls maintained. The GKAP-coated microplate is incubated on a microplate shaker for 90 minutes (300 rpm, 25°C or ambient temperature), followed by washing with TBS-T using the microplate washer. Primary Antibody Solution is added to all GKAP-coated wells, followed by incubation on a microplate shaker for 60 minutes (300 rpm, 25°C or ambient temperature), and washing with TBS-T using the microplate washer. Next, Secondary Antibody Solution is added to all GKAP-coated wells, followed by incubation on a microplate shaker for 60 minutes (300 rpm, 25°C or ambient temperature), and washing with TBS-T using the microplate washer. QuantaBlu Working Solution is then added to all GKAP-coated wells and the microplate is incubated at ambient temperature for 30 minutes prior to adding the QuantaBlu Stop Solution. The assay plate is read using the microplate reader.

[0236] The SoftMax software automatically performs the following calculations. The average RFU for the three non-transduced wells serves as the plate blank and the value is automatically subtracted from the RFU for all dilutions of the reference standard and samples. The software then transforms the florescence data by taking the square root of each result. The parallel-line analysis (PLA) software is then used to determine the percent sample and 95% confidence interval (based on ANOVA error).

[0237] For system suitability, the R2of the reference standard line, as determined by PLA, must be > 0.99100. Additionally, at least 17 / 18 reference standard replicates must have blank adjusted, transformed RFU values greater than 0.

[0238] For sample suitability, the R2of the sample line, as determined by PLA, must be > 0.99100. Additionally, at least 17 / 18 sample replicates must have blank adjusted, transformed RFU values greater than 0. The ratio of parameter estimates must be > 0.56598 and < 1.59238.

[0239] Results are reported as percent relative potency and 95% confidence interval percent, recorded as percentages (%) to three decimal places. If the sample potency is below 50.000%, the result is reported as “Below LOQ, < 50.000%.” If the sample potency is above 140.000%, the result is reported as “Above LOQ, > 140.000%.” The final result is reported according to the release specification (0 decimal places).Example 12: Assay Qualification

[0240] The results obtained from the potency assay qualification are summarized inTable 18. A detailed discussion of the results for each qualification parameter is provided below.Table 18. Summary of ELISA for Functional Activity in JAG201 Transduced Cells QualificationResults

[0241] Accuracy was evaluated by spike recovery studies in which the relative potency (RP) of a spiked sample was compared to the RP of an unspiked sample. Spiked samples were prepared by adding a volume of diluted reference standard equivalent to an RP of 0.2 to JAG201 Pilot Drug Substance Mix samples. Spike recovery was determined in samples diluted to nominal concentrations of 50%, 100%, and 150%. Assays to determine spike recovery at each concentration were performed 3 times (2 runs by Analyst 1 and 1 run by Analyst 2). Spike recoveries for each test session are shown in Table 19.Table 19. Spike Recoveries of 50%, 100%, and 150% Nominal Concentrations

[0242] Precision (repeatability) was evaluated by testing the sample at a 100% nominal concentration in duplicate by the same analyst on the same microplate. Repeatability testing was performed by both Analyst 1 and Analyst 2. The percent difference of the sample tested in sample position 1 and sample position 2, as attained by both analysts, is shown in Table 20. Table 20. Repeatability Results

[0243] Intermediate precision was evaluated by testing the sample at 5 different nominal concentrations. The 50%, 75%, 125%, and 150% nominal concentrations were tested 3 times, whereas the 100% nominal concentration was tested 7 times. The RP attained for each test, the average RP, and %CV for each nominal concentration are shown in Table 21.Table 21. Intermediate Precision Results

[0244] Linearity was evaluated by plotting the observed RP attained for the 5 nominal concentrations, shown in Table 21, versus the expected RP. The resulting trendlines from the linear regression of the data for each of the 3 test sessions are shown in FIG. 16. In addition, the linearity of the data was evaluated by generating a single trendline from the linear regression of the pooled data from the 3 test sessions, which is shown in FIG. 17. The coefficients of determination (R2), slopes, and Y intercepts attained from the resulting trendlines are shown in Table 22.Table 22. Linearity Results

[0245] Based upon the accuracy, precision, and linearity data detailed above, the range of the assay will be set to a RP of > 50% and a RP of < 140%. The data supporting this range are summarized in Table 23.Table 23. Summary of Results Supporting Assay Range

[0246] To confirm specificity of the potency assay, an AAV9 vector containing the SYN-RPL22 genome was tested in the assay. The SYN-RPL22 genome contains the synapsin Ipromoter, which is the same promoter used in construction of the JAG201 genome, with the RLP22 gene instead of the mini SHANK3 gene. As shown in Fig. 18A, increasing doses of JAG201 reference standard resulted in an increase of response (fluorescence). In contrast, increasing doses of SYN-RPL22 did not result in an increase of fluorescence. The resulting R2and RP for the SYN-RPL22 sample are shown in Table 24.

[0247] To confirm the stability -indicating properties of the potency assay, JAG201 Pilot Drug Substance Mix was stressed by heating to 75 °C for 5 minutes prior to transduction of cells in the assay. As shown in FIG. 18B, increasing doses of JAG201 reference standard resulted in an increase of fluorescence. In contrast, increasing doses of thermal stressed JAG201 did not result in an increase of fluorescence. The resulting R2and RP for the thermal stressed JAG201 sample are shown in Table 24.Table 24. Specificity and Stability Indication Results

[0248] Table 25 provides a summary of the qualification results.Table 25. Summary of Qualification Results

[0249] The in vitro potency assay described here measures the relative abundance of miniSHANK3 with the capacity to simultaneously bind GKAP and Homer in JAG201 transduced cells. Given the MO A of SHANK3 in neurons, this assay serves as a suitable predictor of JAG201 biological activity.Table 26. Mouse and Human SHANK3 and miniSHANK3 Sequences and VectorSequencesReferencesDrapeau, Elodie et al. “Behavioral Phenotyping of an Improved Mouse Model of Phelan- McDermid Syndrome with a Complete Deletion of the SHANK3 Gene.” eNeuro vol. 5,3 ENEURO.0046-18.2018. 5 Oct. 2018Kim, E et al. “GKAP, a novel synaptic protein that interacts with the guanylate kinase-like domain of the PSD-95 / SAP90 family of channel clustering molecules.” The Journal of cell biology vol. 136,3 (1997): 669-78.Naisbitt, S et al. “Characterization of guanylate kinase-associated protein, a postsynaptic density protein at excitatory synapses that interacts directly with postsynaptic density-95 / synapse- associated protein 90.” The Journal of neuroscience : the official journal of the Society for Neuroscience vol. 17,15 (1997): 5687-96.Tu, J C et al. “Coupling of mGluR / Homer and PSD-95 complexes by the SHANK family of postsynaptic density proteins.” Neuron vol. 23,3 (1999): 583-92.

[0250] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the disclosure, as defined in the following claims.

Claims

CLAIMSWhat is claimed is:

1. A method for quantitating SHANK3 activity, comprising: a) seeding cells onto a cell culture plate, wherein the cells are inactivated for SHANK3; b) contacting the cells with a sample comprising an AAV vector for a period sufficient for the AAV vector to transduce the cells, wherein the AAV vector comprises an artificial genome comprising a miniSHANK3 transgene; c) lysing the cells to form a lysate comprising miniSHANK3; d) contacting the lysate to a substrate, wherein the substrate comprises a rGKAP peptide; and e) measuring binding activity of SHANK3 or a SHANK3 associated protein to the rGKAP peptide. .

2. The method of claim 1, wherein the method is for quantifying miniSHANK3, the rGKAP peptide is conjugated to biotin.

3. The method of claim 2, wherein the rGKAP peptide is conjugated to biotin at the N- terminus by an SS linker.

4. The method of any one of claims 1 to 3, wherein the rGKAP peptide comprises NSATESAESIEIYIPEAQTRL (SEQ ID NO. 23).

5. The method of any one of claims 1 to 4, wherein the rGKAP peptide is Biotin - SS - NSATESAESIEIYIPEAQTRL (SEQ ID NO. 24).

6. The method of any one of claims 1 to 5, wherein measuring binding activity comprises (1) detecting the binding of the SHANK3 or SHANK3 associated protein to the rGKAP peptide with an anti-SHANK3 or anti-Homer antibody which recognizes the binding of SHANK3 to the rGKAP peptide; (2) detecting the anti-SHANK3 or anti-Homer antibodywith a secondary antibody having a detectable label and (3) measuring a signal from the secondary antibody.

7. The method of claim 6, wherein the anti-SHANK3 antibody is Santa Cruz antibody SC- 377470.

8. The method of claim 7, wherein the secondary antibody is anti-mouse IgG HRP (ThermoFisher A16072).

9. The method of claim 6, wherein the anti-Homer antibody is Abeam ab97593.

10. The method of claim 9, wherein the secondary antibody is goat anti -rabbit IgG HRP (Thermofisher, #G21234).

11. The method of claim 9 or claim 10, wherein the Abeam ab97593 antibody is used at dilutions greater than 1 / 100.

12. The method of any one of claims 6 to 11, wherein the cells are seeded at a density of about le4 to about 4e4 per well and wherein the cells are contacted with the AAV vector in an amount of about lelO vg to about 4el 1 vector genomes (vg) per well.

13. The method of any one of claims 6 through 12, wherein the detectable label is a fluorophore.

14. The method of any one of claims 6 through 12, wherein the detectable label is an enzyme.

15. The method of claim 14, wherein the enzyme is horseradish peroxidase (HRP).

16. The method of any one of claims 6 to 15, wherein the signal is produced by interaction of the detectable label and a detector molecule.

17. The method of claim 16, wherein the detector molecule is 3, 3-, 5,5- Tetranmethylbenzidine (TMB).

18. The method of claim 16, wherein the detector molecule is QuantaBlu.

19. The method of any one of claims 2 to 11, wherein measuring binding activity of SHANK3 or SHANK3 associated proteins to the rGKAP peptide comprises measuring HRP activity with QuantaBlu.

20. The method of any one of claims 1 to 19, wherein the substrate is a microplate.

21. The method of any one of claims 1 to 20, wherein the substrate is a neutravidin coated microplate.

22. The method of any one of claims 1 to 21, wherein the substrate is coated with at least 10 ng rGKAP peptide.

23. The method of claim 22, wherein the substrate is coated with at least 100 ng rGKAP peptide.

24. The method of any one of claims 1 to 23, wherein the cell culture lysate is in a CLBII buffer.

25. The method of any one of claims 1 to 24, wherein step b is performed in the presence of about 4 mM to about 40 mM hydroxyurea.

26. The method of any one of claims 1 to 25, wherein miniSHANK3 has an amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 12 or wherein the transgene comprises a nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 21.

27. The method of any one of claims 1 to 26, wherein the AAV vectors is an AAV9.

28. The method of any one of claims 1 to 27, wherein the cells are human cells.

29. The method of any one of claims 1 to 28, wherein the cells are derived from U-87 MG cells.

30. The method of any one of claims 1 to 29, wherein the period sufficient for the AAV vector to transduce the cells is about 48 to about 96 hours.

31. The method of claim 30, wherein the period sufficient for the AAV vector to transduce the cells is about 96 hours.

32. The method of any one of claims 1 to 31, wherein the assay has a precision (repeatability) of from about 5% to about 13% in identical samples.

33. A method for quantitating miniSHANK3 binding activity, comprising: a) seeding cells onto a cell culture plate, wherein the cells contain a deletion of SHANK3; b) contacting the cells with a sample comprising an AAV vector for a period sufficient for the AAV vector to transduce the cells, wherein the AAV vector comprises an artificial genome comprising a miniSHANK3 transgene; c) lysing the cells to form a lysate comprising miniSHANK3; d) contacting a neutravidin-coated microplate with rGKAP peptides, wherein the rGKAP peptides are conjugated to a biotin at the N terminus with an SS linker, and wherein rGKAP peptides bind to the microplate; and e) washing the neutravidin-coated microplate; f) contacting the rGKAP peptides with the lysate; g) washing the neutravidin-coated microplate; h) contacting the biological sample with an anti-Homer antibody, wherein the anti-Homer antibody is Abeam ab97593; i) washing the neutravidin-coated microplate; j) contacting the anti-Homer antibody with an HRP-conjugated secondary antibody which recognizes the anti-Homer antibody; k) washing the neutravidin-coated microplate;1) contacting the HRP-conjugated secondary antibody with QuantaBlu; and measuring the absorbance at 420nm.

34. The method of claim 33, wherein the assay quantifying for miniSHANK3 has a precision (repeatability) of from about 5% to about 13% in identical samples.

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