Clarification methods

Abstract

The present invention provides a method for clarifying a cell culture, lysate or supernatant comprising AAV3 vector particles, the method comprising: (a) providing a cell culture, lysate or supernatant comprising AAV3 vector particles; and (b) filtering the cell culture, lysate or supernatant through a depth filter to provide a clarified cell culture, lysate or supernatant, wherein the depth filter comprises a first filtration medium comprising an anion exchange nonwoven substrate comprising a plurality of quaternary ammonium groups.

Description

[0001] CLARIFICATION METHODS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to methods for clarifying a cell culture, lysate or supernatant comprising AAV3 vector particles. By AAV3 vector particles, we mean AAV3A, AAV3B, AAV3-like vector particles, and variants thereof. The present invention also relates to a clarified cell culture, lysate or supernatant obtained or obtainable by said methods.

[0004] BACKGROUND TO THE INVENTION

[0005] Adeno-associated virus (AAV) vectors have emerged as a leading platform for gene delivery for treating various diseases due to an excellent safety profile and efficient transduction to various target tissues. However, the large-scale production and longterm storage of AAV vector particles is typically inefficient, resulting in lower yields, moderate purity, and shorter shelf-life compared to recombinant protein therapeutics (see Srivastava, A. , et al. , 2021 . Journal of Pharmaceutical Sciences, 110(7), pp.2609- 2624).

[0006] The production of AAV vector particles typically occurs in cell culture followed by downstream processing. However, efficient downstream processing of AAV vector particles remains a major challenge as AAV serotypes differ in physicochemical properties, making it difficult to design uniform purification processes (see e.g. Meiemeks, F., et al., 2023. Journal of Biotechnology, 367, pp.31 -41 ).

[0007] Clarification of the cell culture, lysate or supernatant is an especially critical step in downstream processing of AAV vector particles. Filtration is often the method of choice for clarification, since it is usually the most straightforward and economical process, and is generally performed using depth filters as a two-stage filtration (see e.g. Meiemeks, F., et al., 2023. Journal of Biotechnology, 367, pp.31 -41 ). However, depth filters come with different media and pore sizes and often involve pre-treatment, multiple time-consuming and expensive further purification steps, posing challenges in achieving high purity and yield of AAV vector particles. Of the AAV serotypes used in gene delivery, AAV3 vectors such as AAV-LK03 have proven to be efficient in targeting human hepatocytes and podocytes (see e.g. Ling, C., et al., 2016. Molecular Therapy Methods & Clinical Development, 3, p.16029; and Ding, W.Y., 2023. Science Translational Medicine, 15(708), p.eabc8226).

[0008] Thus, there is a demand for improved methods for clarifying a cell culture, lysate or supernatant comprising AAV3 vector particles.

[0009] SUMMARY OF THE INVENTION

[0010] The present inventors have developed a method for clarifying a cell culture, lysate or supernatant comprising AAV3 vector particles using a depth filter comprising an anion exchange nonwoven substrate comprising a plurality of quaternary ammonium groups.

[0011] The present inventors have surprisingly shown that using such a depth filter provides improved purity and recovery of AAV3 vector particles compared to other depth filters. The present inventors have also surprisingly shown that using such a depth filter is associated with simplified downstream processing, easy scalability, and time and cost savings.

[0012] In one aspect, the present invention provides a method for clarifying a cell culture, lysate or supernatant comprising AAV3 vector particles, the method comprising (a) providing a cell culture, lysate or supernatant comprising AAV3 vector particles; and (b) filtering the cell culture, lysate or supernatant through a depth filter to provide a clarified cell culture, lysate or supernatant, wherein the depth filter comprises a first filtration medium comprising an anion exchange nonwoven substrate comprising a plurality of quaternary ammonium groups. In some embodiments, the method comprises filtering through a first depth filter. In some embodiments, a second depth filter is used in combination with the first depth filter. In some embodiments, the method comprises the further step of: c) filtering the clarified cell culture, lysate or supernatant from step b) through a second depth filter to provide a further clarified cell culture, lysate or supernatant, optionally wherein the second depth filter comprises a first filtration medium comprising an anion exchange nonwoven substrate comprising a plurality of quaternary ammonium groups.

[0013] In another aspect, the present invention provides a clarified cell culture, lysate or supernatant obtained or obtainable by a method of the present invention.

[0014] In another aspect, the present invention provides a method for providing a composition comprising isolated AAV3 vector particles, the method comprising: (a) providing a cell culture, lysate or supernatant comprising AAV3 vector particles; (b) clarifying the cell culture, lysate or supernatant by a method according to the present invention; and (c) isolating the AAV3 vector particles from the clarified cell culture, lysate or supernatant to provide a composition comprising isolated AAV3 vector particles.

[0015] In another aspect, the present invention provides a composition comprising isolated AAV vector particles obtained or obtainable by a method of the present invention.

[0016] In another aspect, the present invention provides a method for providing a pharmaceutical composition comprising AAV3 vector particles, the method comprising: (a) providing a cell culture, lysate or supernatant comprising AAV3 vector particles; (b) clarifying the cell culture, lysate or supernatant by a method according to the present invention; (c) isolating the AAV3 vector particles from the clarified cell culture, lysate or supernatant to provide a composition comprising isolated AAV3 vector particles; and (d) formulating the composition comprising isolated AAV3 vector particles with one or more pharmaceutically acceptable carrier, diluent and / or excipient to provide a pharmaceutical composition comprising AAV3 vector particles.

[0017] In another aspect, the present invention provides a pharmaceutical composition comprising isolated AAV vector particles obtained or obtainable by a method of the present invention.

[0018] In another aspect, the present invention provides use of a depth filter for clarifying a cell culture, lysate or supernatant comprising AAV3 vector particles, wherein the depth filter comprises a first filtration medium comprising an anion exchange nonwoven substrate comprising a plurality of quaternary ammonium groups. The depth filter may comprise a fluid inlet, a fluid outlet, and one or more filtration mediums fluidly connecting the fluid inlet and the fluid outlet and contained in the same housing. In some embodiments, the depth filter comprises a first filtration medium comprising an anion exchange nonwoven substrate comprising a plurality of quaternary ammonium groups and a second filtration medium comprising a microporous membrane, wherein the first filtration medium is positioned upstream of the second filtration medium. In some embodiments, the depth filter comprises one or more layers, two or more layers, three or more layers, or four or more layers of the first filtration medium.

[0019] The first filtration medium may comprise at least 0.1 mmol of quaternary ammonium groups per gram of the first filtration medium. In some embodiments, the quaternary ammonium groups have the formula -N+R1R2R3X wherein each of R1, R2, and R3are selected from methyl, ethyl, propyl and butyl groups, and wherein X’ is Cl’. In some embodiments, the quaternary ammonium groups are grafted via a linking group directly onto the nonwoven substrate. In some embodiments, the first filtration medium comprises polypropylene nonwovens that have been surface functionalized with a covalently attached quaternary ammonium polymer. In some embodiments, the first filtration medium has a thickness of from 0.1 mm to 10 mm. In some embodiments, the anion exchange nonwoven substrate has an effective fibre diameter of from 1 micrometer to 6 micrometers.

[0020] The microporous membrane may have a mean flow pore size of from 0.1 micrometer to 5 micrometers. In some embodiments, the microporous membrane has an asymmetric pore structure. In some embodiments, the microporous membrane is from 5 micrometers to 800 micrometers thick. In some embodiments, the microporous membrane is formed from polyethersulfone.

[0021] The depth filter may further comprise a membrane support. In some embodiments, the membrane support is formed from polypropylene nonwoven.

[0022] Any suitable filtering conditions may be used. Suitably, the filtering of the cell culture, lysate or supernatant through the depth filter is performed at a flux rate of from 1 L / hr / m2to 300 L / hr / m2, or about 100 L / hr / m2Suitably, the filtering of the cell culture, lysate or supernatant through the depth filter is performed with a differential pressure of from 0.1 bar to 3.0 bar, or from 0.1 bar to 1.5 bar. Suitably, the filtering of the cell culture, lysate or supernatant through the depth filter is performed with a feed volume of from 0.1 L to 5000L, about 200L, or about 500L. Suitably, the filtering of the cell culture, lysate or supernatant through the depth filter is performed at a temperature of from 2°C to 40°C, or from 15°C to 40°C.

[0023] The AAV3 vector particles may be isolated by one or more of tangential flow filtration, normal flow filtration, affinity chromatography, size exclusion chromatography, ion exchange chromatography, mixed mode chromatography and hydrophobic interaction chromatography. In some embodiments, the AAV3 vector particles are isolated by tangential flow filtration and / or affinity chromatography. In some embodiments, the AAV3 vector particles are isolated by tangential flow filtration. In some embodiments, the AAV3 vector particles are isolated by affinity chromatography.

[0024] The methods of the present invention may further comprise any other additional steps. In some embodiments, the method further comprises a step of separating empty AAV3 capsids from full AAV3 capsids. In some embodiments, the method further comprises one or more steps of sterile filtration. In some embodiments, the method further comprises a step (e) of aseptic filling the pharmaceutical composition into a sterile container and sealing the container.

[0025] The AAV3 vector particles may comprise an AAV capsid protein having at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: #1# or SEQ ID NO: #2#. In some embodiments, the AAV3 vector particles comprise an AAV-LK03 capsid protein. In some embodiments, the AAV3 vector particles an AAV capsid protein having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: #3#. In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having at least 99.0%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity to the amino acid sequence of SEQ ID NO: #3#. In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having the amino acid sequence of SEQ ID NO: #3#. The AAV3 vector particles may comprise an AAV genome that is a derivative of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In some embodiments, the AAV genome is a derivative of AAV2. The AAV3 vector particles may encode a therapeutic gene product. In some embodiments, the AAV3 vector particles encode a polypeptide associated with a kidney disease. In some embodiments, the AAV3 vector particles encode a NPHS2, CFI, CFH, FHL-1 , COL4A3, COL4A4, COL4A5, C1 INH, C4BP, MASP2, C3, C5aR1 , C5, C5a, CD55, CD35, CD46, CD59, vitronectin, clusterin, ADCK4, ALG1 , ARHGAP24, ARGHDIA, CD151 , CD2AP, COQ2, COQ6, DGKE, E2F3, EMP2, KANK2, LAGE3, LMNA, LMX1 B, MAF B, NUP85, NUP93, NXF5, OSGEP, PAX2, PDSS2, PMM2, PODXL, SCARB2, SGPL1 , Smad7, TP53RK, TPRKB, VDR, WDR73, WT1 , ZMPSTE24, APOL1 , NPHS1 , TRPC6, NUP107, NUP133, NUP160, ACTN4, INF2, ANKFY1 , ANLN, CRB2, ITGA3, KANK1 , KANK4, MAGI2, MY01 E, OCRL, PTPRO, SMARCAL1 , SYNPO, TBC1 D8B, XPO5, TNS2, NLRP3, or VEGFC polypeptide.

[0026] The cell culture, lysate or supernatant may comprise the AAV3 vector particles in an amount of from 1x1011capsids / mL to 5x1013capsids / mL, optionally determined by ELISA. The cell culture, lysate or supernatant may comprise the AAV3 vector particles in an amount of from 1x1010vg / mL to 5x1012vg / mL, optionally determined by ddPCR.

[0027] In some embodiments, the cell culture, lysate or supernatant is a cell culture. The cell culture may comprise AAV producer and / or packaging cells. In some embodiments, the AAV producer and / or packaging cells are HEK293, HEK293T, COS-1 , COS-7, CV- 1 , HeLa, CHO, SF-9 or A549 cells, or derivatives thereof. In some embodiments, the AAV producer and / or packaging cells are HEK293 cells or derivatives thereof. In some embodiments, the cell culture has a cell density of from 1x106cells / mL to 5x107cells / mL.

[0028] In some embodiments, the cell culture, lysate or supernatant is a cell lysate. The cell lysate may comprise lysed AAV producer and / or packaging cells. In some embodiments, the AAV producer and / or packaging cells are HEK293, HEK293T, COS-1 , COS-7, CV-1 , HeLa, CHO, SF-9 or A549 cells, or derivatives thereof. In some embodiments, the AAV producer and / or packaging cells are HEK293 cells or derivatives thereof. In some embodiments, the cell lysate is derived from a cell culture that has a cell density before lysing of from 1x106cells / mL to 5x107cells / mL. In some embodiments, the cell culture, lysate or supernatant is a cell supernatant.

[0029] The clarified cell culture, lysate or supernatant may have a turbidity of 50 NTU or less, optionally wherein the turbidity is determined by a turbidimeter. The clarified cell culture, lysate or supernatant may have a lower turbidity than the unclarified cell culture, lysate or supernatant. The clarified cell culture, lysate or supernatant may have a dsDNA concentration of 0.5 mg / L or less, 0.4 mg / L or less, 0.3 mg / L or less, 0.2 mg / L or less, or 0.1 mg / L or less, optionally wherein the dsDNA concentration is determined based on the fluorescence intensity of fluorescent dye binding to dsDNA using a fluorometer. The clarified cell culture, lysate or supernatant may comprise the AAV3 vector particles in an amount of from 1x1011capsids / mL to 5x1013capsids / mL, optionally determined by ELISA. The clarified cell culture, lysate or supernatant may have a capsid recovery rate of the AAV3 vector particles of 80% or more, optionally determined by ELISA. The clarified cell culture, lysate or supernatant may comprise the AAV3 vector particles in an amount of from 1x101° vg / mL to 5x1012vg / mL, optionally determined by ddPCR. The clarified cell culture, lysate or supernatant may have a vector genome recovery rate of the AAV3 vector particles of 80% or more, optionally determined by ddPCR.

[0030] In some embodiments, the cell culture, lysate or supernatant is not treated with endonuclease. In some embodiments, the method does not comprise any other clarification steps.

[0031] DESCRIPTION OF DRAWINGS

[0032] Figure 1 - Chromatograms showing pre-column pressure in affinity chromatography

[0033] Chromatogram showing pre-column pressure on affinity purification of filtrate as the load volume after clarification by: (A) a fibrous anion exchange depth filter; (B) GF plus depth filter; (C) PDP8+V100P depth filters; and (D) C0SP depth filter.

[0034] Figure 2 - Sequence alignment of AAV3 VP1 capsid proteins

[0035] Sequence alignment performed by Clustal Omega for AAV3B, AAV3A, AAV-LK03, AAV3B-DE5, AAV.GT5, AAV3-ST, AAV3B-V04, AAV3B-V05, AAV3B-G3, and AAV3B-E12 VP1 capsid proteins. The AAV3 VP1 capsid proteins share at least about 95% sequence identity.

[0036] DETAILED DESCRIPTION

[0037] Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples. This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. The skilled person will understand that they can combine all features of the invention disclosed herein without departing from the scope of the invention as disclosed.

[0038] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0039] The terms "comprising", "comprises" and "comprised of' as used herein are synonymous with "including", "includes", "containing", or "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or steps. The terms "comprising", "comprises" and "comprised of" also include the term "consisting of".

[0040] Numeric ranges are inclusive of the numbers defining the range. As used herein the term “about” means approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical value or range, it modifies that value or range by extending the boundaries above and below the numerical value(s) set forth. In general, the terms “about” and “approximately” may be used herein to modify a numerical value(s) above and below the stated value(s) by 10%.

[0041] Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation and any amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0042] All publications mentioned in the specification are herein incorporated by reference. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0043] Methods for clarifying AAV3 vector particles

[0044] The present invention provides a method for clarifying a cell culture, lysate or supernatant lysate comprising AAV3 vector particles, the method comprising: (a) providing a cell culture, lysate or supernatant comprising AAV3 vector particles; and (b) filtering the cell culture, lysate or supernatant through a depth filter to provide a clarified cell culture, lysate or supernatant.

[0045] The present invention also provides a clarified cell culture, lysate or supernatant obtained or obtainable by said method.

[0046] The methods of the present invention may be good manufacturing practice (GMP)- compatible methods. Good manufacturing practice (GMP) describes the minimum standard that a medicines manufacturer must meet in their production processes (see e.g. World Health Organization, 2007. Quality assurance of pharmaceuticals: a compendium of guidelines and related materials. Good manufacturing practices and inspection (Vol. 2)).

[0047] In some embodiments, the methods of the present invention include upstream processing of the cell culture, lysate or supernatant. For example, methods of the present invention may include treatment with nuclease or endonuclease, addition of salt, pH adjustment, and / or addition of a flocculent, or any combination thereof.

[0048] In some embodiments, the upstream processing does not include centrifugation of the cell culture, lysate or supernatant. In some embodiments, the upstream processing does not include filtration of the cell culture, lysate or supernatant. In some embodiments, the upstream processing does not include centrifugation or filtration of the cell culture, lysate or supernatant.

[0049] In some embodiments, the upstream processing does not include treatment with nuclease. In some embodiments, the upstream processing does not include treatment with endonuclease. In some embodiments, the upstream processing does not include treatment with nuclease or endonuclease. In some embodiments, the upstream processing does not include centrifugation and / or filtration of the cell culture, lysate or supernatant and does not include treatment with nuclease and / or endonuclease. In some embodiments, the upstream processing does not include centrifugation or filtration of the cell culture, lysate or supernatant and does not include treatment with nuclease or endonuclease.

[0050] In some embodiments, the methods of the present invention do not include upstream processing of the cell culture, lysate or supernatant.

[0051] In some embodiments, the method does not comprise any other clarification steps. In some embodiments, the method does not comprise any other filtration steps during clarification. In some embodiments, the method does not comprise filtering through any other depth filter.

[0052] Alternatively, in some embodiments, the method does comprise filtering through a second depth filter in combination with the first depth filter. For example, in some embodiments, the method comprises a further step of: c) filtering the clarified cell culture, lysate or supernatant from step b) through a second depth filter to provide a further clarified cell culture, lysate or supernatant. In some embodiments, the second depth filter comprises a first filtration medium comprising an anion exchange nonwoven substrate comprising a plurality of quaternary ammonium groups.

[0053] The second depth filter may be the same or different to the first depth filter. The second depth filter may be the same as the first depth filter. The second filter may be different to the first depth filter. In some embodiments, the first and second depth filters are both anion exchange depth filters e.g. fibrous anion exchange filters such as the 3M Harvest RC depth filter or the 3M Harvest RC Centrate depth filter. For example, in some embodiments, the first depth filter is the 3M Harvest RC depth filter and the second depth filter is the 3M Harvest RC Centrate depth filter, or vice versa. The first and second depth filters may have a different pore size at entry to the filter.

[0054] In some embodiments, the method does not comprise a step of acidifying the clarified cell culture, lysate or supernatant. In some embodiments, the method does not comprise a step of binding the AAV3 vector particles to the depth filter(s) and / or does not comprise a step of eluting the AAV3 vector particles from the depth filter(s). Cell culture, lysate or supernatant.

[0055] The method of the present invention comprises a step of providing a cell culture, lysate or supernatant comprising AAV3 vector particles.

[0056] Methods of preparing and modifying AAV vector particles, are well known in the art. For example, suitable methods are described in Ayuso, E., et al., 2010. Current gene therapy, 10(6), pp.423-43. AAV3 vector particles may be produced by introducing a plasmid encoding an AAV genome and one or more helper plasmids encoding AAV replication and AAV3 capsid proteins into a cell line.

[0057] The concentration of AAV3 vector particles in the cell culture, lysate or supernatant may be determined by any suitable method in the art, for example by AAV capsid ELISA and / or by a quantitative PCR method, such as ddPCR or qPCR (see e.g. Smith, P.H., et al., 2003. Molecular Therapy, 7(1 ), pp.122-128).

[0058] In some embodiments, the cell culture, lysate or supernatant comprises the AAV3 vector particles in an amount of at least about 1x1010capsids / mL, at least about 5x1010capsids / mL, at least about 1x1011capsids / mL, or at least about 5x1011capsids / mL. In some embodiments, the cell culture, lysate or supernatant comprises the AAV3 vector particles in an amount of about 5x1014capsids / mL or less, about 1x1014capsids / mL or less, about 5x1013capsids / mL or less, or about 1x1013capsids / mL or less. In some embodiments, the cell culture, lysate or supernatant comprises the AAV3 vector particles in an amount of from about 1x101° capsids / mL to about 5x1014capsids / mL, from about 5x1010capsids / mL to about 1x1014capsids / mL, or from about 1x1011capsids / mL to about 5x1013capsids / mL. The concentration of capsids may be determined by AAV capsid ELISA (see e.g. Kuck, D., et al., 2007. Journal of virological methods, 140(1 -2), pp.17-24).

[0059] In some embodiments, the cell culture, lysate or supernatant comprises the AAV3 vector particles in an amount of at least about 1x109vg / mL, at least about 5x109vg / mL, at least about 1x101° vg / mL, or at least about 5x101° vg / mL. In some embodiments, the cell culture, lysate or supernatant comprises the AAV3 vector particles in an amount of about 5x1013vg / mL or less, about 1x1013vg / mL or less, about 5x1012vg / mL or less, or about 1x1012vg / mL or less. In some embodiments, the cell culture, lysate or supernatant comprises the AAV3 vector particles in an amount of from about 1x109vg / mL to about 5x1013vg / mL, from about 5x109vg / mL to about 1x1013vg / mL, or from about 1x1010vg / mL to about 5x1012vg / mL. The concentration of vector genomes may be determined by ddPCR (see e.g. Dobnik, D., et al., 2019. Frontiers in microbiology, 10, p.429957).

[0060] AA V producer and / or packaging cells

[0061] The AAV3 vector particles may be produced and / or packaged in AAV producer and / or packaging cells and the cell culture, lysate or supernatant may be derived therefrom.

[0062] The term “AAV producer cell” includes a cell that produces AAV vector particles, after transient transfection, stable transfection or vector transduction of all the elements necessary to produce the AAV vector particles, or any cell engineered to stably comprise the elements necessary to produce the AAV vector particles. Suitable AAV producer cells will be known to those of skill in the art (see e.g. Martin, J., et al. 2013. Human gene therapy methods, 24(4), pp.253-269) and may include HEK293, COS-1 , COS-7, CV-1 , HeLa, CHO, and A549 cells.

[0063] The term “AAV packaging cell” includes a cell which contains some or all of the elements necessary for packaging a recombinant AAV genome. Typically, such packaging cells contain one or more vectors which are capable of expressing AAV structural proteins (e.g. AAV rep and cap genes) and / or one or more genes encoding the AAV structural proteins have been integrated into the genome of the packaging cell. Cells comprising only some of the elements required for the production of AAV vector particles are useful as intermediate reagents in the generation of AAV vector particle producer cell lines, through subsequent steps of transient transfection, transduction or stable integration of each additional required element. These intermediate reagents are encompassed by the term “packaging cell”. Suitable AAV packaging cells will be known to those of skill in the art (see e.g. Martin, J., et al. 2013. Human gene therapy methods, 24(4), pp.253-269).

[0064] In some embodiments, the AAV producer and / or packaging cells are HEK293, HEK293T, COS-1 , COS-7, CV-1 , HeLa, CHO, SF-9 or A549 cells, or derivatives thereof. In some embodiments, the AAV producer and / or packaging cells are HEK293 cells, or a derivative thereof (e.g. a HEK293T cell, a HEK293T-Rex cell, a HEK293FT cell, a HEK293SF-3F6 cell, a HEK293SF-3F9 cell, a HEK293-EBNA1 cell, or a SJ293TS cell).

[0065] In some embodiments, the AAV producer and / or packaging cells are HEK293 cells. In some embodiments, the AAV producer and / or packaging cells are HEK293T cells.

[0066] Cell culture

[0067] In some embodiments, the cell culture, lysate or supernatant is a cell culture.

[0068] As used herein, a “cell culture” may refer to a culture of cells which have been grown and maintained under controlled conditions. In addition to the AAV3 vector particles, a “cell culture” may comprise intact cells, cell debris, and colloids.

[0069] Typically, the cells are derived from multicellular eukaryotes, for example animal cells. In some embodiments, the cell culture is a mammalian cell culture. In some embodiments, the cell culture is a human cell culture. In some embodiments, the cell culture is a HEK293 cell culture.

[0070] In some embodiments, the cell culture comprises AAV producer and / or packaging cells. In some embodiments, the cell culture has a cell density of at least about 1x105cells / mL, at least about 5x105cells / mL, or at least about 1x106cells / mL. In some embodiments, the cell culture has a cell density of about 1x108cells / mL or less, about 5x107cells / mL or less, about 1x107cells / mL or less, or about 5x106cells / mL or less. In some embodiments, the cell culture has a cell density of from about 1x105cells / mL to about 1x108cells / mL, from about 5x105cells / mL to about 5x107cells / mL, from about 1x106cells / mL to about 5x107cells / mL, or from about 1x106cells / mL to about 1x107cells / mL.

[0071] Cell lysate

[0072] In some embodiments, the cell culture, lysate or supernatant is a cell lysate.

[0073] As used herein, a “cell lysate” may refer to the preparation obtained after lysing cells in a cell culture. During cell lysis the cell membrane is broken down or destroyed to release AAV viral particles from the host cell. A “cell lysate” may be substantially free of intact cells. Suitable methods of lysing cells are also known in the art and include, for example, multiple freeze / thaw cycles, sonication, microfluidization, and treatment with chemicals (e.g. detergents) (see e.g. Srivastava, A., et al. 2022, Pharmaceutical Technology, pp.38-43).

[0074] In some embodiments, the cell lysate is a mammalian cell lysate. In some embodiments, the cell lysate is a human cell lysate. In some embodiments, the cell lysate is a HEK293 cell lysate.

[0075] In some embodiments, the cell lysate comprises lysed AAV producer and / or packaging cells. In some embodiments, the cell lysate is derived from a cell culture that has a cell density before lysing of at least about 1x105cells / mL, at least about 5x105cells / mL, or at least about 1x106cells / mL. In some embodiments, the cell lysate is derived from a cell culture that has a cell density before lysing of about 1x108cells / mL or less, about 5x107cells / mL or less, about 1x107cells / mL or less, or about 5x106cells / mL or less. In some embodiments, the cell lysate is derived from a cell culture that has a cell density before lysing of from about 1x105cells / mL to about 1x108cells / mL, from about 5x105cells / mL to about 5x107cells / mL, from about 1x106cells / mL to about 5x107cells / mL, or from about 1x106cells / mL to about 1x107cells / mL.

[0076] Cell culture supernatant

[0077] In some embodiments, the cell culture, lysate or supernatant is a cell culture supernatant.

[0078] As used herein, a “cell culture supernatant” may refer to the media in which cells were growing in a cell culture. A “cell culture supernatant” may be substantially free of intact cells and cell debris and, for example, may be obtained by centrifuging a cell culture or cell lysate.

[0079] In some embodiments, the cell culture supernatant is a mammalian cell culture supernatant. In some embodiments, the cell culture supernatant is a human cell culture supernatant. In some embodiments, the cell culture supernatant is a HEK293 cell culture supernatant. In some embodiments, the cell culture supernatant is derived from a cell culture comprising AAV producer and / or packaging cells or a cell lysate comprising lysed AAV producer and / or packaging cells. In some embodiments, the cell culture supernatant is derived from a cell culture that has a cell density of at least about 1x105cells / mL, at least about 5x105cells / mL, or at least about 1x106cells / mL. In some embodiments, the cell culture supernatant is derived from a cell culture that has a cell density of about 1x108cells / mL or less, about 5x107cells / mL or less, about 1x107cells / mL or less, or about 5x106cells / mL or less. In some embodiments, the cell culture supernatant is derived from a cell culture that has a cell density of from about 1x105cells / mL to about 1x108cells / mL, from about 5x105cells / mL to about 5x107cells / mL, from about 1x106cells / mL to about 5x107cells / mL, or from about 1x106cells / mL to about 1x107cells / mL.

[0080] Depth filters

[0081] The method of the present invention comprises a step of filtering the cell culture, lysate or supernatant through a depth filter to provide a clarified cell culture, lysate or supernatant.

[0082] As used herein, a “depth filter” may refer to a filter that uses one or more filtration mediums to retain particles. A depth filter may entrap or adsorb particles within and on the filter due to a random matrix or structure that creates a tortuous path through the filter.

[0083] The depth filter used in the present invention may comprise any suitable structure. In some embodiments, the depth filter comprises a fluid inlet, a fluid outlet, and one or more filtration mediums fluidly connecting the fluid inlet and the fluid outlet. In preferred embodiments, the one or more filtration mediums are contained in the same housing. In other embodiments, the one or more filtration mediums are contained in separate housings.

[0084] The depth filter used in the present invention may be referred to as a “fibrous anion exchange depth filter”. In some embodiments, the depth filter comprises a filtration medium comprising an anion exchange nonwoven substrate. In some embodiments, the depth filter comprises a first filtration medium comprising an anion exchange nonwoven substrate and a second filtration medium comprising a microporous membrane. In some embodiments, the first filtration medium is positioned upstream of the second filtration medium.

[0085] In some embodiments, depth filter comprises a housing which has a top wall, a bottom wall, a generally cylindrical side wall extending between the top wall and the bottom wall, a filter element disposed within the housing comprising an inlet and an outlet defining a liquid flow path through the filter element. The liquid flow path may have an upstream portion adjacent the inlet and a downstream portion adjacent the outlet so that the fluid flows through the first filtration medium and then through the second filtration medium to the outlet.

[0086] In some embodiments, the first and second filtration mediums may be each individually configured as a planar or lenticular disk. In some embodiments, the first and second filtration mediums may be each individually pleated. Preferably, the first and second filtration mediums are located in close proximity, more preferably the first filtration medium and the second filtration medium are housed within the same filter housing. In some embodiments, the first and second filtration mediums are configured as a planar or lenticular disk. In some embodiments, the first and second filtration mediums are pleated together. Examples of pleating configurations and filter devices comprising pleated media may be found, for example, in U.S. Pat. No. 6,315,130 and U.S. Pat. No. 6,521 ,011 . In some embodiments, the second filtration medium is wrapped around a core and the first filtration medium is wrapped about the second filtration medium. Alternatively, the second filtration medium may be disposed onto the first filtration medium to form a multi-layered construction, wherein a single layer of the multilayered construction is wrapped around the circumference of a core and bonded together parallel to the core axis, wherein the second filtration medium is downstream from the first filtration medium. Examples of lenticular filter cells and methods of making lenticular filter cells may be found, for example, in U.S. Pat. No. 6,464,084, U.S. Pat. No. 6,939,466, U.S. Pat. No. 7,178,676, and U.S. Pat. No. 6,712,966, and in U.S. Pat. No. 10,918,985.

[0087] In some embodiments, the filter device further comprises a separator element, wherein the separator element comprises a central core in fluid communication with the fluid inlet; a first side; and a second side; and an edge seal; wherein the filter media further comprises a first media disk positioned on the first side of the separator element and having an outer circumferential edge and an inner circumferential edge; and a second media disk positioned on the second side of the separator element and having an outer circumferential edge and an inner circumferential edge; and wherein the outer circumferential edges of the first and second media disks are connected by the edge seal and the inner circumferential edges of the first and second media disks are connected to the central core and wherein the upstream side of the first and second media disk comprises the first filtration medium.

[0088] An example depth filter for use in the present invention is the 3M Harvest RC depth filter. The 3M Harvest RC is a depth filter containing multiple layers of quaternary amine functional anion exchange nonwoven media and a microporous membrane for fine particulate removal (see e.g. Almeida, A., et al., 2022. Biotechnology Progress, 38(2), p.e3227).

[0089] Another example depth filter for use in the present invention is the 3M Harvest RC Centrate depth filter. The 3M Harvest RC Centrate is a fibrous anion exchange depth filter comprising multiple layers of quaternary amine functional anion exchange nonwoven media, a microporous asymmetric polyamide membrane functionalised with quaternary ammonium nominally rated at 0.2 pm for fine particulate removal and a non-functionalized polypropylene nonwoven used as a support layer.

[0090] Previously, the 3M Harvest RC Centrate depth filter is known in the art to be used after centrifugation has taken place, and the 3M Harvest RC Centrate and 3M Harvest RC depth filters are not meant to be used in the same purification sequence, see https: / / multimedia.3m.com / mws / media / 2415861 O / 3m-harvest-rc-centrate- chromatographic-clarifier-product-information-sheet.pdf?&fn=harvest-rc-centrate- product-info-sheet-pf-bf-en.pdf. However, the inventors have surprisingly found that the 3M Harvest Centrate RC depth filter can be used in combination with the 3M Harvest RC depth filter to filter / clarify AAV3 vector particles by further reducing turbidity. When these two filters are used in combination, with the 3M Harvest RC depth filter being the first depth filter and the 3M Harvest Centrate being the second depth filter, the 3M Harvest RC depth filter is being used in place of centrifugation. This is a new use for the 3M Harvest RC depth filter.

[0091] First filtration medium The first filtration medium may be any suitable anion exchange nonwoven substrate.

[0092] As used herein, the term "nonwoven substrate" or “nonwoven web” may refer to a fabric that has a structure of individual fibers or filaments which are randomly and / or unidirectionally interlaid in a mat-like fashion (see e.g. Lavoie, J., et al., 2023. Biotechnology and Bioengineering, pp 1 -18).

[0093] The nonwoven substrate may be manufactured using any technique known in the art. For example, the nonwoven substrate can be made by carded, air laid, wet laid, spunlaced, spunbonding, electrospinning or melt-blowing techniques, such as melt- spun or meltblown, or combinations thereof. Staple fibers may also be present in the nonwoven substrate. The presence of staple fibers generally provides a loftier, less dense web than a web of only melt blown microfibers. Preferably, no more than about 20 weight percent staple fibers are present, more preferably no more than about 10 weight percent staple fibers are present.

[0094] The nonwoven substrate may be made from a single type of fiber or two or more fibers that differ in the type of thermoplastic polymer and / or thickness. The nonwoven substrate may be formed from fibers or filaments made of any suitable thermoplastic polymeric material. Suitable polymeric materials include, but are not limited to, polyolefins, poly(isoprenes), poly(butadienes), fluorinated polymers, chlorinated polymers, polyamides, polyimides, polyethers, poly(ether sulfones), poly(sulfones), poly(vinyl acetates), copolymers of vinyl acetate, such as poly(ethylene)-co-poly(vinyl alcohol), poly(phosphazenes), poly(vinyl esters), poly(vinyl ethers), poly(vinyl alcohols), and poly(carbonates). Suitable polyolefins include, but are not limited to, poly(ethylene), poly(propylene), poly(1 -butene), copolymers of ethylene and propylene, alpha olefin copolymers (such as copolymers of ethylene or propylene with 1 -butene, 1 -hexene, 1 -octene, and 1 -decene), poly(ethylene-co-1 -butene) and poly(ethylene-co-1 -butene-co-1 -hexene). Suitable fluorinated polymers include, but are not limited to, poly(vinyl fluoride), poly(vinylidene fluoride), copolymers of vinylidene fluoride (such as poly(vinylidene fluoride-co-hexafluoropropylene), and copolymers of chlorotrifluorocthylene (such as poly(cthylene-co- chlorotrifluoroethylene). Suitable polyamides include, but are not limited to: poly(iminoadipoyliminohexamethylene), poly(iminoadipoyliminodecamethylene), and polycaprolactam. Suitable polyimides include poly(pyromellitimide). Suitable poly(ether sulfones) include, but are not limited to, poly(diphenylether sulfone) and poly(diphenylsulfone-co-diphenylene oxide sulfone). Suitable copolymers of vinyl acetate include, but are not limited to, poly(ethylene-co-vinyl acetate) and such copolymers in which at least some of the acetate groups have been hydrolyzed to afford various poly(vinyl alcohols) including, poly(ethylene-co-vinyl alcohol).

[0095] In some embodiments, the nonwoven substrate is made from one or more polyolefins. In some embodiments, the nonwoven substrate is made from polypropylene.

[0096] The effective fiber diameter may be calculated according to the method set forth in Davies, C. N., "The Separation of Airborne Dust and Particles," Institution of Mechanical Engineers, London, Proceedings IB, 1952. In some embodiments, the microfibers of the nonwoven substrate have an effective fiber diameter of at least about 0.5 micrometers, at least about 1 micrometer, at least about 2 micrometers, or at least about 4 micrometers. In some embodiments, the microfibers of the nonwoven substrate have an effective fiber diameter of at most about 15 micrometers, at most about 10 micrometers, at most about 8 micrometers, or at most about 6 micrometers. In some embodiments, the microfibers of the nonwoven substrate have an effective fiber diameter of from about 0.5 micrometers to about 15 micrometers, from about 1 micrometer to about 10 micrometers, from about 1 micrometers to about 8 micrometers, or from about 1 micrometers to about 6 micrometers.

[0097] In some embodiments, the nonwoven substrate has a basis weight of at least about 5 g / m2, at least about 10 g / m2, at least about 20 g / m2, or at least about 50 g / m2. In some embodiments, the nonwoven substrate has a basis weight of at most about 1000 g / m2, at most about 800 g / m2, at most about 600 g / m2, at most about 500 g / m2, at most about 400 g / m2, at most about 200 g / m2, or at most about 100 g / m2. In some embodiments, the nonwoven substrate has a basis weight of from about 5 g / m2to about 1000 g / m2, from about 10 g / m2to about 500 g / m2from about 10 g / m2to about 200 g / m2, or from about 10 g / m2to about 100 g / m2.

[0098] The minimum tensile strength of the nonwoven substrate is typically about 4.0 Newtons. The nonwoven substrate typically has a solidity of about 20% or less, or about 15% or less. Nonwoven substrate loft may be measured by solidity, a parameter that defines the solids fraction in a volume of substrate. The nonwoven substrate may optionally further comprise one or more layers of scrim. For example, either or both major surfaces may each optionally further comprise a scrim layer. The scrim, which is typically a woven or nonwoven reinforcement made from fibers, is included to provide strength to the nonwoven article. Suitable scrim materials include, but are not limited to, nylon, polyester, fiberglass, polyethylene, polypropylene, and the like. The average thickness of the scrim can vary. Typically, the average thickness of the scrim ranges from about 25 to about 100 micrometers, preferably about 25 to about 50 micrometers. The layer of the scrim may optionally be bonded to the nonwoven substrate. A variety of adhesive materials can be used to bond the scrim to the nonwoven substrate. Alternatively, the scrim may be heat- bonded to the nonwoven substrate.

[0099] The nonwoven substrate may be treated to comprise quaternary ammonium functional groups. In some embodiments, the first filtration medium is an anion exchange nonwoven substrate comprising quaternary ammonium groups.

[0100] In some embodiments, the first filtration medium comprises at least about 0.1 mmol, at least about 0.2 mmol, at least about 0.4 mmol, at least about 0.8 mmol, at least about 1 mmol, or at least about 5 mmol of quaternary ammonium groups per gram of the first filtration medium.

[0101] In some embodiments, the quaternary ammonium groups have the formula - N+R1R2R3X’. In some embodiments, each of R1, R2, and R3are alkyl groups. In some embodiments, each of R1, R2, and R3are alkyl groups having from 1 to 20, from 1 to 10, from 1 to 6, or from 1 to 4 carbon atoms. In some embodiments, each of R1, R2, and R3are selected from methyl, ethyl, propyl and butyl groups. In preferred embodiments, each of R1, R2, and R3are methyl groups. In other embodiments, one of R1, R2, and R3is methyl and the other two are an alkyl having from 2 to 18, from 2 to 10, from 2 to 6, or from 2 to 4 carbon atoms. In other embodiments, two of R1, R2, and R3are methyl and the other is an alkyl having from 2 to 18, from 2 to 10, from 2 to 6, or from 2 to 4 carbon atoms. In yet other embodiments, at least two of R1, R2, and R3combine with the nitrogen atom to which they are attached to form a heterocyclic group. The heterocyclic group may include at least one nitrogen atom and can contain other heteroatoms such as oxygen or sulfur. Exemplary heterocyclic groups include, but are not limited to, piperidinyl and morpholinyl. The heterocyclic group can be fused to an additional ring such as a benzene, cyclohexene, or cyclohexane. X’ may be any suitable counter ion. In some embodiments, X’ is a halide, a sulfate, a phosphate, or a nitrate. In some embodiments, X’ is a halide. In some embodiments, X’ is Cl’.

[0102] The quaternary ammonium groups may be covalently bonded to the nonwoven substrate using any technique known in the art. For example, the manufacture of anion exchange nonwovens is described in U.S. Pat. No. 8,328,023. Typically, the quaternary ammonium groups are grafted via a linking group directly onto the nonwoven substrate or onto a nonwoven substrate that has been treated with a primer layer. In some embodiments, the nonwoven substrate is grafted using a quaternary ammonium salt of an aminoalkyl(meth)acryloyl monomer. Exemplary quaternary salts of include, but are not limited to, (meth)acrylamidoalkyltrimethylammonium salts and (meth)acryloxyalkyltrimethyl-ammonium salts. Monomers having a quaternary ammonium group may be directly grafted to the surface of the nonwoven substrate, or a aminoalkyl (meth)acryloyl monomer, having a primary, secondary or tertiary amine group, may be grafted and subsequently converted to a quaternary ammonium group by alkylation. More information regarding such a grafting may be found in U.S. Pat. No. 9,821 ,276.

[0103] In some embodiments, the grafted polymers comprising the quaternary ammonium group are polymers derived from aminoalkyl (meth)acryloyl monomer, poly(alkylene oxide) monomer and optionally a second hydrophilic monomer. In some embodiments, the first filtration medium comprises a polymer grafted to the surface of a nonwoven substrate wherein the polymer comprises interpolymerized monomeric units of: (a) from 80 to 98 wt% of an aminoalkyl(meth)acryloyl monomer; (b) from 2 to 20 wt% of a poly(alkylene oxide) monomer; and (c) from 0 to 10 wt% of a second hydrophilic monomer. Examples of suitable poly(alkylene oxide) monomers include poly(ethylene oxide) (meth)acrylate, polypropylene oxide) (meth)acrylate, poly(ethylene oxidepropylene oxide) (meth)acrylate, and combinations thereof. Examples of suitable hydrophilic monomers include 2-hydroxyethyl(meth)acrylate (HEMA), 2- hydroxypropyl(meth)acrylate, 3-hydroxypropyl(meth)acrylate, 2,3-dihydroxypropyl (meth)acrylate, 4-hydroxybutyl(meth)acrylate, N-vinyl caprolactam, N-vinyl acetamide, N-vinyl pyrrolidone, acrylonitrile, tetrahydrofurfuryl acrylate, acrylamide, mono- or di-N-alkyl substituted acrylamide, glycerol methacrylate, and combinations thereof.

[0104] The first filtration medium may be any suitable thickness. In some embodiments, the thickness of the first filtration medium is at least about 0.1 mm, at least about 0.25 mm, or at least about 1 mm. In some embodiments, the thickness of the first filtration medium is at most about 10 mm, at most about 8 mm, or at most about 5 mm. In some embodiments, the thickness of the first filtration medium is from about 0.1 mm to about 10 mm, from about 0.25 mm to about 8 mm, or from about 1 mm to about 5 mm.

[0105] In some embodiments, the depth filter comprises one or more layers of the first filtration medium. In some embodiments, the depth filter comprises one or more layers, two or more layers, three or more layers, or four or more layers of the first filtration medium. In some embodiments, the depth filter comprises four layers of the first filtration medium. When a plurality of first filtration medium layers is used, each layer may have the same, or different effective fiber diameter, basis weight, solidity, amount of quaternary ammonium grafted, and / or thickness. In some embodiments, each subsequent layer of the first filtration medium may have a smaller effective fiber diameter so that finer contaminants may be retained.

[0106] Previously, it is expected that AAV particles will bind to quaternary ammonium groups within the depth filter, meaning said AAV particles do not flow through and are trapped by the filter. Surprisingly, the inventors have found that when using a depth filter comprising a first filtration medium having quaternary ammonium groups, AAV3 vector particle(s) within a cell culture, lysate or supernatant do not bind to quaternary ammonium groups. Therefore, AAV3 vector particle(s) can pass through a first filtration medium comprising quaternary ammonium groups.

[0107] Second filtration medium

[0108] The second filtration medium may be any suitable microporous membrane.

[0109] As used herein, a “microporous membrane” may refer to a structure having a morphology of a precisely controlled interconnected network of pores typically ranging from about 0.03 pm up to about 10 pm in diameter and an otherwise impermeable polymeric structure. The microporous membrane may be manufactured using any technique known in the art. In some embodiments, the microporous membrane is a solvent-induced phase separation (SIPS) membrane. In other embodiments, the microporous membrane is a thermally-induced phase separation (TIPS) membrane. In terms of membrane geometry, three types of microporous membranes are commercially available: flat sheet, hollow fiber, and tubular membrane.

[0110] The microporous membrane may be a porous polymeric substrate (such as sheet or film) comprising micropores with a mean flow pore size of about 5 micrometers or less. In some embodiments, the microporous membrane has a mean flow pore size of at least about 0.1 micrometers, at least about 0.2 micrometers, at least about 0.5 micrometers, at least about 0.8 micrometers, or at least about 1 micrometer. In some embodiments, the microporous membrane has a mean flow pore size of at most about 4 micrometers, at most about 3 micrometers, or at most about 2 micrometers. In some embodiments, the microporous membrane has a mean flow pore size of from about 0.1 micrometers to about 5 micrometers, from about 0.2 micrometers to about 4 micrometers, from about 0.2 micrometers to about 3 micrometers, or from about 0.2 micrometer to about 2 micrometers. In some embodiments, the microporous membrane has a mean flow pore size of about 0.2 micrometers. The mean flow pore size may be determined by ASTM Standard Test Method No. F316-03.

[0111] The microporous membrane can have a symmetric or asymmetric (e.g., gradient) distribution of pore size in the direction of fluid flow. In some embodiments, the microporous membrane has an asymmetric pore structure.

[0112] The microporous membrane may be formed from any suitable thermoplastic polymeric material. Suitable polymeric materials include, but are not limited to, polyolefins, poly(isoprenes), poly(butadienes), fluorinated polymers, chlorinated polymers, polyamides, polyimides, polyethers, poly(ether sulfones), poly(sulfones), poly(vinyl acetates), polyesters such as poly(lactic acid), copolymers of vinyl acetate, such as poly(ethylene)-co-poly(vinyl alcohol), poly(phosphazenes), poly(vinyl esters), poly(vinyl ethers), poly(vinyl alcohols), and poly(carbonates). Suitable polyolefins include, but are not limited to, poly(ethylene), poly(propylene), poly(1 -butene), copolymers of ethylene and propylene, alpha olefin copolymers (such as copolymers of ethylene or propylene with 1 -butene, 1 -hexene, 1 -octene, and 1 -decene), poly(ethylene-co-1 -butene) and poly(ethylene-co-1 -butene-co-1 -hexene). Suitable fluorinated polymers include, but are not limited to, poly(vinyl fluoride), poly(vinylidene fluoride), copolymers of vinylidene fluoride (such as poly(vinylidene fluoride-co- hexafluoropropylene), and copolymers of chlorotrifluoroethylene (such as poly(ethylene-co-chlorotrifluoroethylene). Suitable polyamides include, but are not limited to, poly(iminoadipolyliminohexamethylene), poly(iminoadipolyliminodecamethylene), and polycaprolactam. Suitable polyimides include, but are not limited to, poly(pyromellitimide). Suitable poly(ether sulfones) include, but are not limited to, poly(diphenylether sulfone) and poly(diphenylsulfone- co-diphenylene oxide sulfone). Suitable copolymers of vinyl acetate include, but are not limited to, poly(ethylene-co-vinyl acetate) and such copolymers in which at least some of the acetate groups have been hydrolyzed to afford various poly(vinyl alcohols).

[0113] In some embodiments, the microporous membrane is formed from poly(ether sulfone).

[0114] In some embodiments, the microporous membrane is treated to comprise a functional group, such as a guanidyl functional group. More information regarding such functionalisation may be found in U.S. Pat. No. 10,722,848. Alternatively, the microporous membrane does not comprise a plurality of guanidyl groups.

[0115] In some embodiments, AAV3 vector particles do not bind to the microporous membrane.

[0116] The second filtration medium may be any suitable thickness. In some embodiments, the thickness of the second filtration medium is at least about 5 micrometers, at least about 10 micrometers, at least about 20 micrometers, at least about 25 micrometers, or at least about 50 micrometers. In some embodiments, the thickness of the second filtration medium is at most about 1000 micrometers, at most about 800 micrometers, at most about 500 micrometers, at most about 200 micrometers, or at most about 100 micrometers. In some embodiments, the thickness of the second filtration medium is from about 5 micrometers to about 1000 micrometers, from about 5 micrometers to about 800 micrometers, from about 10 micrometers to about 500 micrometers, from about 20 micrometers to about 200 micrometers, or from about 25 micrometers to about 1000 micrometers. In some embodiments, the depth filter comprises one or more layers of the second filtration medium. In some embodiments, the depth filter comprises one layer of the second filtration medium. When a plurality of the second filtration medium is used, each layer may have a symmetric or asymmetric (e.g., gradient) distribution of pore size through the direction of fluid flow, and the layers may have the same, or different mean flow pore size and / or thickness. In some embodiments, each subsequent layer of the second filtration medium may have a smaller mean flow pore size so that finer contaminants may be filtered.

[0117] Membrane support

[0118] The depth filter may further comprise a membrane support.

[0119] In some embodiments, the first and second filtration mediums are encapsulated with a nonwoven substrate to provide support and aid in handling. In filtration applications, the first and second filtration mediums may be disposed either vertically, or horizontally in the membrane support.

[0120] The membrane support may be formed from fibers or filaments made of any suitable thermoplastic polymeric material described above. Suitable polymeric materials include, but are not limited to, polyolefins, poly(isoprenes), poly(butadienes), fluorinated polymers, chlorinated polymers, polyamides, polyimides, polyethers, poly(ether sulfones), poly(sulfones), poly(vinyl acetates), copolymers of vinyl acetate, such as poly(ethylene)-co-poly(vinyl alcohol), poly(phosphazenes), poly(vinyl esters), poly(vinyl ethers), poly(vinyl alcohols), and poly(carbonates).

[0121] In some embodiments, the membrane support is made from one or more polyolefins. In some embodiments, the membrane support is made from polypropylene.

[0122] Filtering conditions

[0123] Any suitable conditions may be used when filtering the cell culture, lysate or supernatant through the depth filter. Suitable operating conditions are provided by depth filter manufacturers and the skilled person will be able to optimise the conditions depending on the depth filter design and scale, to achieve the desired clarification and throughput. The cell culture, lysate or supernatant may be filtered through the depth filter using any suitable flux rate. The flux rate may be about 500 L / hr / m2or less, about 400 L / hr / m2or less, about 300 L / hr / m2or less, about 200 L / hr / m2or less, or about 100 L / hr / m2or less. The flux rate may be about 1 L / hr / m2or more, about 5 L / hr / m2or more, about 10 L / hr / m2or more, about 50 L / hr / m2or more, or about 100 L / hr / m2or more. In some embodiments, the flux rate is from about 1 L / hr / m2to about 500 L / hr / m2, from about 1 L / hr / m2to about 400 L / hr / m2, from about 1 L / hr / m2to about 300 L / hr / m2, from about 5 L / hr / m2to about 200 L / hr / m2, from about 10 L / hr / m2to about 200 L / hr / m2, or from about 50 L / hr / m2to about 200 L / hr / m2. In some embodiments, the flux rate is about 100 L / hr / m2.

[0124] The cell culture, lysate or supernatant may be filtered through the depth filter using any suitable inlet pressure. The inlet pressure may be about 5.0 bar or less, about 4.0 bar or less, about 3.9 bar or less, about 3.8 bar or less, about 3.7 bar or less, about 3.6 bar or less, about 3.5 bar or less, about 3.4 bar or less, about 3.3 bar or less, about 3.2 bar or less, about 3.1 bar or less, about 3.0 bar or less, about 2.9 bar or less, or about 2.8 bar or less. The inlet pressure may be about 0.1 bar or more, about 0.5 or more, or about 1 .0 bar or more. In some embodiments, the inlet pressure is from about 0.1 bar to about 5.0 bar, from about 0.1 bar to about 4.0 bar, from about 0.1 bar to about 3.4 bar, from about 0.1 bar to about 3.1 bar, or from about 0.1 bar to about 2.8 bar.

[0125] The cell culture, lysate or supernatant may be filtered through the depth filter using any suitable differential pressure. The differential pressure may be about 3.0 bar or less, about 2.9 bar or less, about 2.8 bar or less, about 2.7 bar or less, about 2.6 bar or less, about 2.5 bar or less, about 2.4 bar or less, about 2.3 bar or less, about 2.2 bar or less, about 2.1 bar or less, about 2.0 bar or less, about 1.9 bar or less, about 1 .8 bar or less, about 1 .7 bar or less, about 1 .6 bar or less, or about 1 .5 bar or less. The differential pressure may be about 0.1 bar or more, about 0.2 bar or more, about 0.3 bar or more, about 0.4 bar or more, or about 0.5 or more. In some embodiments, the differential pressure is from about 0.1 bar to about 3.0 bar, from about 0.1 bar to about 2.5 bar, from about 0.1 bar to about 2.0 bar, or from about 0.1 bar to about 1 .5 bar. The cell culture, lysate or supernatant may be filtered through the depth filter using any suitable feed volume. The feed volume may be about 10000L or less, about 5000L or less, about 4000L or less, about 3000L or less, about 2000L or less, about WOOL or less, or about 500L or less. The feed volume may be about 0.1 L or more, about 1 L or more, about 10L or more, about 100L or more, or about 200L or more. In some embodiments, the feed volume is from about 0.1 L to about 10000L, from about 0.1 L to 5000L, from about 0.1 L to about WOOL, from about 1 L to about WOOL, from about 10L to about WOOL, from about 100L to about WOOL, or from about 200L to about 500L. In some embodiments, the feed volume is about 200L. In some embodiments, the feed volume is about 500L.

[0126] The cell culture, lysate or supernatant may be filtered through the depth filter using any suitable temperature. The temperature may be about 50°C or less, about 45°C or less, about 40°C or less, about 35°C or less, about 30°C or less, or about 25°C or less. The temperature may be about 1 °C or more, about 2°C or more, about 3°C or more, about 4°C or more, about 5°C or more, about 10°C or more, or about 15°C or more. In some embodiments, the temperature is from about 1 °C to about 50°C, from about 2°C to about 40°C, from about 5°C to about 40°C, from about 10°C to about 40°C, or from about 15°C to about 40°C.

[0127] The method may comprise a preconditioning flush prior to contact of the depth filter with the cell culture, lysate or supernatant. The required minimum preconditioning flush is typically provided by the depth filter manufacturer. The preconditioning flush may be equivalent to 25 L / m2at a flux of 300 L / hr / m2

[0128] Clarified cell culture, lysate or supernatant

[0129] The present inventors have surprisingly found that the clarification method of the present invention is sufficient to provide a clarified cell culture, lysate or supernatant with high purity and recovery, without requiring further clarification steps or filtering. For example, unlike prior art methods which use other depth filters, the present inventors have surprisingly found that no acidification is needed to eliminate impurities which remain in the clarified cell culture, lysate or supernatant.

[0130] The purity of the clarified cell culture, lysate or supernatant may be determined by measuring the turbidity of the clarified cell culture, lysate or supernatant in an acidification assay. In the acidification assay, the turbidity of the clarified cell culture, lysate or supernatant may be determined following acidification. The clarified cell culture, lysate or supernatant may be acidified by addition of 1 M acetic acid. The turbidity may be determined by a turbidimeter. The turbidity may be expressed in Nephelometric Turbidity Units (NTU), for example defined according to USEPA Method 180.1. The clarified cell culture, lysate or supernatant may have a turbidity of about 100 NTU or less, about 80 NTU or less, or about 50 NTU or less upon acidification. The clarified cell culture, lysate or supernatant may have a lower turbidity than the unclarified cell culture, lysate or supernatant.

[0131] The purity of the clarified cell culture, lysate or supernatant may additionally or alternatively be determined by measuring the concentration of dsDNA in the clarified cell culture, lysate or supernatant. The clarified cell culture, lysate or supernatant may have a dsDNA concentration of about 1 .0 mg / L or less, about 0.9 mg / L or less, about 0.8 mg / L or less, about 0.7 mg / L or less, about 0.6 mg / L or less, about 0.5 mg / L or less, about 0.4 mg / L or less, about 0.3 mg / L or less, about 0.2 mg / L or less, or about 0.1 mg / L or less. The dsDNA concentration may be determined based on the fluorescence intensity of fluorescent dye binding to dsDNA using a fluorometer. For example, suitable fluorescent DNA-binding dyes include PicoGreen (see e.g. Ahn, S.J., et al., 1996. Nucleic acids research, 24(13), pp.2623-2625).

[0132] The clarified cell culture, lysate or supernatant may have a capsid recovery rate of the AAV3 vector particles of about 50% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 100%. In some embodiments, the clarified cell culture, lysate or supernatant comprises the AAV3 vector particles in an amount of at least about 1x1010capsids / mL, at least about 5x1010capsids / mL, at least about 1x1011capsids / mL, or at least about 5x1011capsids / mL. In some embodiments, the clarified cell culture, lysate or supernatant comprises the AAV3 vector particles in an amount of about 5x1014capsids / mL or less, about 1x1014capsids / mL or less, about 5x1013capsids / mL or less, or about 1x1013capsids / mL or less. In some embodiments, the clarified cell culture, lysate or supernatant comprises the AAV3 vector particles in an amount of from about 1x1010capsids / mL to about 5x1014capsids / mL, from about 5x1010capsids / mL to about 1x1014capsids / mL, or from about 1x1011capsids / mL to about 5x1013capsids / mL. The concentration of capsids may be determined by AAV capsid ELISA (see e.g. Kuck, D., et al., 2007. Journal of virological methods, 140(1- 2), pp.17-24).

[0133] The clarified cell culture, lysate or supernatant may have a vector genome recovery rate of the AAV3 vector particles of about 50% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 100%. In some embodiments, the clarified cell culture, lysate or supernatant comprises the AAV3 vector particles in an amount of at least about 1x109vg / mL, at least about 5x109vg / mL, at least about 1x101° vg / mL, or at least about 5x101° vg / mL. In some embodiments, the clarified cell culture, lysate or supernatant comprises the AAV3 vector particles in an amount of about 5x1013vg / mL or less, about 1x1013vg / mL or less, about 5x1012vg / mL or less, or about 1x1012vg / mL or less. In some embodiments, the clarified cell culture, lysate or supernatant comprises the AAV3 vector particles in an amount of from about 1x109vg / mL to about 5x1013vg / mL, from about 5x109vg / mL to about 1x1013vg / mL, or from about 1x1010vg / mL to about 5x1012vg / mL. The concentration of vector genomes may be determined by ddPCR (see e.g. Dobnik, D., et al., 2019. Frontiers in microbiology, 10, p.429957).

[0134] Methods for providing a composition

[0135] The present invention provides a method for providing a composition comprising isolated AAV3 vector particles, the method comprising:

[0136] (a) providing a cell culture, lysate or supernatant comprising AAV3 vector particles;

[0137] (b) filtering the cell culture, lysate or supernatant through a depth filter to provide a clarified cell culture, lysate or supernatant; and

[0138] (c) isolating the AAV3 vector particles from the clarified cell culture, lysate or supernatant to provide a composition comprising isolated AAV3 vector particles. The present invention also provides a composition comprising isolated AAV vector particles obtained or obtainable by said method.

[0139] The steps of providing a cell culture, lysate or supernatant comprising AAV3 vector particles and filtering the cell culture, lysate or supernatant through a depth filter to provide a clarified cell culture, lysate or supernatant may be performed as described above in the section entitled “Methods for clarifying AAV3 vector particles”.

[0140] The AAV3 vector particles may be isolated by any suitable methods known in the art. Suitable methods include tangential flow filtration, normal flow filtration, affinity chromatography, size exclusion chromatography, ion exchange chromatography, mixed mode chromatography and hydrophobic interaction chromatography.

[0141] In some embodiments, the AAV3 vector particles are isolated by one or more, two or more, three or more, or four or more of tangential flow filtration, normal flow filtration, affinity chromatography, size exclusion chromatography, ion exchange chromatography, mixed mode chromatography and hydrophobic interaction chromatography.

[0142] In some embodiments, the AAV3 vector particles are isolated by tangential flow filtration (TFF). As used herein, “tangential flow filtration" or “cross-flow microfiltration” may refer to a mode of filtration in which the solute-containing solution passes tangentially across a membrane and molecules which are larger than the membrane pores are passed through by applying pressure. Filtration-based processes such as tangential flow filtration have commonly been used to isolate AAV vector particles (see e.g. Miyaoka, R., et al., 2023. Biotechnology and Bioengineering, 120(11 ), pp.3311- 3321 ).

[0143] In some embodiments, the AAV3 vector particles are isolated by normal flow filtration. As used herein, “normal flow filtration" or “dead-end filtration” may refer to a mode of filtration in which the solute-containing solution passes vertically through a membrane and molecules which are larger than the membrane pores are pass through.

[0144] In some embodiments, the AAV3 vector particles are isolated by affinity chromatography. As used herein, “affinity chromatography” is a method of separating a biomolecule from a mixture, based on a highly specific macromolecular binding interaction between the biomolecule and another substance. Suitable affinity chromatography media are known in the art and include without limitation, AVB Sepharose™ and POROS™ CaptureSelect™ AAVX affinity resin (see e.g. Mietzsch, M., et al., 2020. Molecular Therapy-Methods & Clinical Development, 19, pp.362-373; and Florea, M., et al., 2023. Molecular Therapy Methods & Clinical Development, 28, pp.146-159).

[0145] In some embodiments, the AAV3 vector particles are isolated by size exclusion chromatography (SXC). As used herein, “size exclusion chromatography” is a method of separating a biomolecule from a mixture based on the size and / or molecular weight of the biomolecule. Suitable size exclusion agents are known in the art and include polyethylene glycol (PEG) (see e.g. Marichal-Gallardo, P., et al., 2021. Human Gene Therapy, 32(17-18), pp.959-974).

[0146] In some embodiments, the AAV3 vector particles are isolated by ion exchange chromatography (IEX). As used herein, “ion exchange chromatography” is a method of separating a biomolecule from a mixture based on the net charge of the biomolecule. Ion exchange chromatography has been used to purify specific AAV serotypes (see e.g. Brument, N., et al., 2002. A Molecular Therapy, 6(5), pp.678-686).

[0147] In some embodiments, the AAV3 vector particles are isolated by hydrophobic interaction chromatography. As used herein, “hydrophobic interaction chromatography” is a method of separating a biomolecule from a mixture based on its hydrophobicity. Hydrophobic interaction chromatography has been used to purify specific AAV serotypes (see e.g. McNally, D.J., et al., 2020. Molecular Therapy- Methods & Clinical Development, 19, pp.275-284).

[0148] In some embodiments, the AAV3 vector particles are isolated by mixed mode chromatography (MMC). As used herein, “mixed mode chromatography” is a method of separating a biomolecule from a mixture based on at least two modes of interaction such as electrostatic and hydrophobic interactions.

[0149] In some embodiments, more than one method is used to isolate the AAV3 vector particles. For example, in some embodiments, the clarified cell culture, lysate or supernatant is concentrated via tangential flow filtration before being applied to a chromatographic medium. Concentration of the clarified cell culture, lysate or supernatant enables a manageable volume of clarified feed to be subjected to chromatography and allows for more reasonable sizing of columns without the need for lengthy recirculation times. In some embodiments, the clarified cell culture, lysate or supernatant is concentrated by at least about two-fold, by at least about ten-fold, by at least about twenty-fold, by at least about fifty-fold, by about 100-fold, or by about 200-fold before being applied to a chromatographic medium.

[0150] In some embodiments, the clarified cell culture, lysate or supernatant or composition comprising isolated AAV3 vector particles is subjected to concentration, for example by tangential flow filtration. In some embodiments, the clarified cell culture, lysate or supernatant or composition comprising isolated AAV3 vector particles is subjected to diafiltration to remove small molecule impurities, for example by tangential flow filtration. In some embodiments, the diafiltration comprises the use of from about 3 to about 10 diafiltration volume of buffer. In some embodiments, the clarified cell culture, lysate or supernatant or composition comprising isolated AAV3 vector particles is subjected to buffer exchange.

[0151] In some embodiments, the AAV3 vector particles are isolated by tangential flow filtration and one or more of affinity chromatography, size exclusion chromatography, ion exchange chromatography, mixed mode chromatography and hydrophobic interaction chromatography. In some embodiments, the AAV3 vector particles are isolated by tangential flow filtration and affinity chromatography. In some embodiments, the AAV3 vector particles are isolated by tangential flow filtration and size exclusion chromatography. In some embodiments, the AAV3 vector particles are isolated by tangential flow filtration and ion exchange chromatography. In some embodiments, the AAV3 vector particles are isolated by tangential flow filtration and mixed mode chromatography. In some embodiments, the AAV3 vector particles are isolated by tangential flow filtration and hydrophobic interaction chromatography.

[0152] In some embodiments, the method further comprises a step of separating empty AAV3 capsids from full AAV3 capsids. Any suitable method may be used to separate empty AAV3 capsids from full AAV3 capsids, for example cesium chloride (CsCI) or iodixanol density gradient centrifugation or ion exchange chromatography. In some embodiments, ion exchange chromatography is used to separate empty AAV3 capsids from full AAV3 capsids. In some embodiments, the method further comprises one or more step of sterile filtration of the clarified cell culture, lysate or supernatant or composition comprising isolated AAV3 vector particles. Any suitable method may be used for sterile filtration. In some embodiments, a 0.2 pm sterilizing grade filter is used.

[0153] Methods for providing a pharmaceutical composition

[0154] The present invention provides a method for providing a pharmaceutical composition comprising AAV3 vector particles, the method comprising:

[0155] (a) providing a cell culture, lysate or supernatant comprising AAV3 vector particles;

[0156] (b) filtering the cell culture, lysate or supernatant through a depth filter to provide a clarified cell culture, lysate or supernatant;

[0157] (c) isolating the AAV3 vector particles from the clarified cell culture, lysate or supernatant to provide a composition comprising isolated AAV3 vector particles; and

[0158] (d) formulating the composition comprising isolated AAV3 vector particles with one or more pharmaceutically acceptable carrier, diluent and / or excipient to provide a pharmaceutical composition comprising AAV3 vector particles.

[0159] The present invention also provides a pharmaceutical composition comprising AAV3 vector particles obtained or obtainable by said method.

[0160] The steps of providing a cell culture, lysate or supernatant comprising AAV3 vector particles and filtering the cell culture, lysate or supernatant through a depth filter to provide a clarified cell culture, lysate or supernatant may be performed as described above in the section entitled “Methods for clarifying AAV3 vector particles”.

[0161] The step of isolating the AAV3 vector particles from the clarified cell culture, lysate or supernatant to provide a composition comprising isolated AAV3 vector particles may be performed as described above in the section entitled “Methods for providing a composition”.

[0162] Pharmaceutical compositions As used herein, a “pharmaceutical composition” may refer to a composition that comprises or consists of a therapeutically effective amount of a pharmaceutically active agent i.e. the AAV3 vector particle. It preferably includes a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof).

[0163] By “pharmaceutically acceptable” is included that the formulation is sterile and pyrogen free. The carrier, diluent, and / or excipient must be “acceptable” in the sense of being compatible with the vector and not deleterious to the recipients thereof. Typically, the carriers, diluents, and excipients will be saline or infusion media which will be sterile and pyrogen free; however, other acceptable carriers, diluents, and excipients may be used.

[0164] Acceptable carriers, diluents, and excipients for therapeutic use are well known in the pharmaceutical art. The choice of pharmaceutically acceptable carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s) or solubilising agent(s).

[0165] Examples of pharmaceutically acceptable carriers include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

[0166] The pharmaceutical composition may be a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solution may be suitably buffered (preferably to a pH of from 3 to 9). The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

[0167] Suitably, the pharmaceutical composition comprises an isotonic buffer (e.g. at about pH 7.4). In some embodiments, the pharmaceutical composition comprises phosphate buffered saline (PBS) buffer (e.g. at about pH 7.4). Optionally the PBS is supplemented with about 200 mM NaCI. In some embodiments, the pharmaceutical composition comprises plasmalyte. In some embodiments, the pharmaceutical composition comprises about 0.001 % poloxamer 188 (also known as Pluronic F-68).

[0168] The pharmaceutical composition may comprise the AAV3 vector particles in any suitable amount. Suitably, the pharmaceutical composition comprises the AAV3 vector particles in an amount of about 1x107vg / ml or more, about 1x108vg / ml or more, about 1x109vg / ml or more, about 1x1010vg / ml or more, about 1x1011vg / ml or more, about 1x1012vg / ml or more, or about 1x1013vg / ml or more. Suitably, the pharmaceutical composition comprises the AAV3 vector particles in an amount of about 1x1014vg / ml or less or about 1x1013vg / ml or less. Suitably, the pharmaceutical composition comprises the AAV3 vector particles in an amount of from about 1x107vg / ml to about 1x1014vg / ml. Suitably, the pharmaceutical composition comprises the AAV3 vector particles in an amount of from about 1x107vg / ml to about 5x1013vg / ml. Suitably, the pharmaceutical composition comprises the AAV3 vector particles in an amount of about 1x107vg / ml to about 10x107vg / ml, about 1x108vg / ml to about 10x108vg / ml, about 1x109vg / ml to about 10x109vg / ml, about 1x1010vg / ml to about 10x1010vg / ml, about 1x1011vg / ml to about 10x1011vg / ml, about 1x1012vg / ml to about 10x1012vg / ml, or about 1x1013vg / ml to about 10x1013vg / ml.

[0169] In some embodiments, the pharmaceutical composition comprises the AAV3 vector particles in an amount of from about 1x1010vg / ml to about 1x1013vg / ml. In some embodiments, the pharmaceutical composition comprises the AAV3 vector particles in an amount of from about 1x101° vg / ml to about 1x1012vg / ml.

[0170] The pharmaceutical composition may be sterilized. In some embodiments, the method further comprises a step of sterile filtration of the pharmaceutical composition. Any suitable method may be used for sterile filtration. In some embodiments, a 0.2 pm sterilizing grade filter is used.

[0171] The pharmaceutical composition may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. In some embodiments, the method further comprises a step of aseptic filling the pharmaceutical composition into a sterile container and sealing the container. The pharmaceutical composition may be any suitable volume. Suitably, the pharmaceutical composition has a volume of from about 5 ml to about 50 ml, from about 5 ml to about 25 ml, or from about 10 ml to about 25 ml. In some embodiments, the pharmaceutical composition has a volume of about 1 ml, about 2 ml, about 3 ml, about 4 ml, about 5 ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, about 10 ml, about 11 ml, about 12 ml, about 13 ml, about 14 ml, about 15 ml, about 16 ml, about 17 ml, about 18 ml, about 19 ml, about 20 ml, about 21 ml, about 22 ml, about 23 ml, about 24 ml, about 25 ml, about 26 ml, about 27 ml, about 28 ml, about 29 ml, or about 30 ml.

[0172] The pharmaceutical composition may further comprise one or more other therapeutic agents.

[0173] AAV vector particles

[0174] An AAV vector particle is encapsidated by capsid proteins and may be referred to in terms of its serotype.

[0175] A serotype corresponds to a variant subspecies of AAV which, owing to its profile of expression of capsid surface antigens, has a distinctive reactivity which can be used to distinguish it from other variant subspecies. Typically, an AAV vector particle having a particular AAV serotype does not efficiently cross-react with neutralising antibodies specific for any other AAV serotype. AAV serotypes include AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11 , and derivatives thereof.

[0176] The serotype may facilitate the transduction of specific cell types. AAV vector particles may be transcapsidated forms wherein an AAV genome or derivative having an ITR of one serotype is packaged in the capsid of a different serotype. The AAV vector particles may be in the form of a pseudotyped AAV vector particle. AAV vector particles may also be chemically modified, bearing ligands adsorbed to the capsid surface. For example, such ligands may include antibodies for targeting a particular cell surface receptor.

[0177] The VP1 , VP2 and VP3 proteins make up the capsid of an AAV particle, which determines the AAV serotype. VP1 , VP2, and VP3 may be produced by alternate mRNA splicing (see e.g. Trempe, J.P. and Carter, B.J., 1988. Journal of virology, 62(9), pp.3356-3363). Thus, VP1 , VP2 and VP3 may have identical sequences, but wherein VP2 is truncated at the N-terminus relative to VP1 , and VP3 is truncated at the N-terminus relative to VP2.

[0178] Generation of the AAV-KP1 , AAV-KP2 and AAV-KP3 vector particles has been described in Pekrun, K., et al., 2019. JCI Insight 2019;4(22):e131610. AAV3B is the parental sequence that is most closely related to AAV-KP1 , AAV-KP2 and AAV-KP3 vector particles; AAV-KP1 and AAV-KP3 demonstrate 92% sequence identity to AAV3B and AAV-KP2 demonstrates 95% identity to AAV3B. The AAV-KP1 capsid sequence contains fragments from at least 7 of the 8 parental serotypes used in the capsid shuffling library whereas AAV-KP2 and AAV-KP3 contained fragments from at least 6 parental serotypes.

[0179] AAV-KP1 , AAV-KP2 and AAV-KP3 vector particles may comprise an AAV-KP1 , AAV- KP2 or AAV-KP3 capsid protein or a variant thereof, respectively. For example, AAV- KP1 vector particles may comprise AAV-KP1 VP1 proteins, AAV-KP1 VP2 proteins, and AAV-KP1 VP3 proteins, or variants thereof. For example, AAV-KP2 vector particles may comprise AAV-KP2 VP1 proteins, AAV-KP2 VP2 proteins, and AAV- KP2 VP3 proteins, or variants thereof. For example, AAV-KP3 vector particles may comprise AAV-KP3 VP1 proteins, AAV-KP3 VP2 proteins, and AAV-KP3 VP3 proteins, or variants thereof. AAV-KP1 , AAV-KP2, and AAV-KP3 vector particles may comprise a total number of 60 VP1 , VP2, and VP3 subunits per capsid. AAV- KP1 , AAV-KP2, and AAV-KP3 vector particles may comprise KP1 , KP2 or KP3 VP1 , VP2 and VP3 capsid proteins, or variants thereof, in a ratio of 1 : 1 : 10 (VP1 :VP2:VP3).

[0180] AAV-KP1 , AAV-KP2 and AAV-KP3 variant capsids have been described in the art (see e.g. Pekrun, K., et al., 2019. JCI Insight 2019;4(22):e131610 and WO 2019 / 191701 A1 ). Further AAV-KP1 , AAV-KP2 and AAV-KP3 variant capsids may be generated by amino acid substitutions, deletions, or insertions.

[0181] An example AAV-KP1 VP1 capsid sequence is provided below in SEQ ID NO: #34# (GenBank accession number MN428626; Protein accession number: QFR04619). AAV-KP1 VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: #34#. An example AAV-KP2 VP1 capsid sequence is provided below in SEQ ID NO: #35# (GenBank accession number MN428627.1 ; Protein accession number QFR04620.1 ). AAV-KP2 VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: #35#.

[0182] An example AAV-KP1 VP3 capsid sequence is provided below in SEQ ID NO: #36# (GenBank accession number MN428628,.1 ; Protein accession number QFR04621 ). AAV-KP3 VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: #36#.

[0183] Methods to generate AAV capsid variants are well known in the art (see e.g. Lee, E. J., et al., 2018. Current opinion in biomedical engineering, 7, pp.58-63). For example, the efficiency and specificity of AAV gene delivery can be improved using point mutations on the viral capsid. A second rational design approach is to introduce functional domains non-viral in nature into the AAV capsid to elicit desired functions. A third rational design approach involves using chemical biology strategies to make more precise modifications to the capsid.

[0184] The sequences of AAV capsid genes may be genetically modified to introduce specific deletions, substitutions or insertions with respect to a native AAV wild-type sequence. In particular, AAV capsid genes may be modified by the insertion of a sequence of an unrelated protein or peptide within an open reading frame of the AAV capsid coding sequence, or at the N- and / or C-terminus of the AAV capsid coding sequence. The unrelated protein or peptide may advantageously be one which acts as a ligand for a particular cell type, thereby conferring improved binding to a target cell or improving the specificity of targeting of the vector to a particular cell population. The unrelated protein may also be one which assists purification of the viral particle as part of the production process, i.e. an epitope or affinity tag. The site of insertion will typically be selected so as not to interfere with other functions of the AAV viral particle e.g. internalisation, trafficking of the AAV viral particle, etc.

[0185] In some embodiments, a capsid protein variant has at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the reference capsid protein. In some embodiments, a capsid protein variant has at least 98.0%, at least 98.1 %, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identity to the reference capsid protein.

[0186] In some embodiments, a capsid protein variant has 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 15 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution compared to the reference capsid protein.

[0187] In some embodiments, a capsid protein variant has 1 substitution, 2 substitutions, 3 substitutions, 4 substitutions, 5 substitutions, 6 substitutions, 7 substitutions, 8 substitutions, 9 substitutions, 10 substitutions, 11 substitutions, 12 substitutions, 13 substitutions, 14 substitutions, 15 substitutions, 16 substitutions, 17 substitutions, 18 substitutions, 19 substitutions, 20 substitutions, 21 substitution, 22 substitutions, 23 substitutions, 24 substitutions, 25 substitutions, 26 substitutions, 27 substitutions, 28 substitutions, 29 substitutions, or 30 substitutions compared to the reference capsid protein.

[0188] In some embodiments, a capsid protein variant has an insertion of 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid compared to the reference capsid protein.

[0189] In some embodiments, a capsid protein variant has an insertion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids compared to the reference capsid protein.

[0190] In some embodiments, a capsid protein variant has a deletion of 10 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid compared to the reference capsid protein. In some embodiments, a capsid protein variant has a deletion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, or 5 amino acids compared to the reference capsid protein.

[0191] In some embodiments, compared to the reference capsid protein, a capsid protein variant has:

[0192] (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution;

[0193] (b) an insertion of 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or les, or 1 amino acid; and / or

[0194] (c) a deletion of 10 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid.

[0195] In some embodiments, compared to the reference capsid protein, a capsid protein variant has:

[0196] (a) 1 substitution, 2 substitutions, 3 substitutions, 4 substitutions, 5 substitutions, 6 substitutions, 7 substitutions, 8 substitutions, 9 substitutions, 10 substitutions, 11 substitutions, 12 substitutions, 13 substitutions, 14 substitutions, 15 substitutions, 16 substitutions, 17 substitutions, 18 substitutions, 19 substitutions, 20 substitutions, 21 substitution, 22 substitutions, 23 substitutions, 24 substitutions, 25 substitutions, 26 substitutions, 27 substitutions, 28 substitutions, 29 substitutions, or 30 substitutions;

[0197] (b) an insertion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids; and / or (c) a deletion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, or 5 amino acids.

[0198] In some embodiments, compared to the reference capsid protein, a capsid protein variant has: (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) no insertions; and (c) no deletions.

[0199] In some embodiments, compared to the reference capsid protein, a capsid protein variant has: (a) no substitutions; (b) an insertion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids; and (c) no deletions.

[0200] In some embodiments, compared to the reference capsid protein, a capsid protein variant has: (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) an insertion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids; and (c) no deletions.

[0201] AAV3 serotype and capsid proteins

[0202] As used herein, the “AAV3 vector particles” may refer to AAV vector particles which have serologic cross-reactivity with the AAV3 serotype.

[0203] AAV serotype 3 is serologically distinct from other serotypes (see e.g. Lerch, T.F., et al., 2010. Virology, 403(1 ), pp.26-36). Serologic cross-reactivity may be determined by any method known in the art (see e.g. Gao, G., et al., 2004. Journal of virology, 78(12), pp.6381 -6388; and Gao, G.P., et al., 2002. PNAS, 99(18), pp.11854-11859). For example, antisera to the AAV3 serotype may be generated by injection of AAV3 vector particles. The AAV vector particles may be pre-incubated with the AAV3 antiserum, and then incubated with indicator 84-31 cells for 48 to 72 h. Transduction may be assessed under a UV microscope. Neutralizing antibody titers may be reported as the highest serum dilution that inhibited transduction by 50% of that seen with serum from a naive animal.

[0204] In some embodiments, AAV3 vector particles have a neutralization titer of 1 / 80 or less, 1 / 160 or less, 1 / 320 or less, 1 / 640 or less, 1 / 1280 or less, 1 / 2560 or less, 1 / 5120 or less, 1 / 10240 or less, 1 / 20480 or less, or 1 / 40960 or less when incubated with AAV3 antiserum. In some embodiments, AAV3 vector particles have a neutralization titer of 1 / 5120 or less, 1 / 10240 or less, 1 / 20480 or less, or 1 / 40960 or less when incubated with AAV3 antiserum. In some embodiments, AAV3 vector particles have a neutralization titer of about 1 / 5120, about 1 / 10240, about 1 / 20480, or about 1 / 40960 when incubated with AAV3 antiserum.

[0205] AAV3 was originally isolated from humans and may use heparan sulfate proteoglycan (HSPG), human fibroblast growth factor receptor 1 (FGFR1 ), laminin receptor (LR) and / or the human hepatocyte growth factor receptor (HGFR) for cell entry (see Issa, S.S., et al., 2023. Cells, 12(5), p.785; Akache, B., et al., 2006. Journal of virology, 80(19), pp.9831 -9836; Ling, C., et al., 2010. Human gene therapy, 21 (12), pp.1741 - 1747; and Messina, E.L., et al., 2012. Human gene therapy, 23(10), pp.1031 -1042).

[0206] In some embodiments, AAV3 vector particles bind one or more of HSPG, FGFR1 , LR, and HGFR. In some embodiments, AAV3 vector particles bind each of HSPG, FGFR1 , LR, and HGFR. Binding affinity to the receptors may be determined by any method known in the ar. For example, by using affinity chromatography with affinity columns comprising HSPG, FGFR1 , LR, or HGFR (see e.g. Cabanes-Creus, M., et al., 2021. Molecular Therapy Methods & Clinical Development, 21 , pp.607-620).

[0207] AAV3 vector particles may be capable of transducing hepatocytes and / or podocytes (see e.g. Ling, C., et al., 2016. Molecular Therapy Methods & Clinical Development, 3, p.16029; and Ding, W.Y., 2023. Science Translational Medicine, 15(708), p.eabc8226).

[0208] In some embodiments, AAV3 vector particles are capable of transducing hepatocytes. Transduction efficiency of hepatocytes may be determined by any method known in the art (see e.g. Ling, C., et al., 2016. Molecular Therapy Methods & Clinical Development, 3, p.16029). For example, by measuring the expression of a reporter transgene, e.g. GFP, carried by the AAV3 vector particle, wherein expression of the transgene in hepatocytes correlates with the ability of the AAV vector particle to transduce hepatocytes.

[0209] In some embodiments, AAV3 vector particles are capable of transducing podocytes. Transduction efficiency of podocytes may be determined by any method known in the art (see e.g. Ding, W.Y., 2023. Science Translational Medicine, 15(708), p.eabc8226). For example, by measuring the expression of a reporter transgene, e.g. GFP, carried by the AAV3 vector particle, wherein expression of the transgene in podocytes correlates with the ability of the AAV vector particle to transduce podocytes.

[0210] AAV3 vector particles may comprise an AAV3 capsid protein or a variant thereof. For example, AAV3 vector particles may comprise AAV3 VP1 proteins, AAV3 VP2 proteins, and AAV3 VP3 proteins, or variants thereof.

[0211] AAV3 variant capsids have been described in the art (see e.g. WO 2024 / 015877, WO 2021 / 108468, WO 2017 / 070476, and WO 2014 / 193716). Moreover, it has been shown that site-directed mutagenesis of specific surface-exposed amino acids and amino acid insertions can significantly increase the transduction efficiency. For example, it has been shown that site-directed mutagenesis of surface exposed tyrosine to phenylalanine residues on AAV3 capsids significantly improves the transduction efficiency of Y701 F, Y705F and Y731 F mutant AAV3 vectors and the Y705+731 F double-mutant AAV3 vector (see e.g. Cheng, B., et al., 2012. Gene therapy, 19(4), pp.375-384); S663V+T492V double-mutant AAV3 vectors had increased in transduction livers (see e.g. Ling, C., et al., 2016. Molecular Therapy Methods & Clinical Development, 3, p.16029); transduction efficiency of Y705+731 F and S663V+T492V+K533R mutants was increased by ~ 10-fold and that of S663V+T492V mutants was increased by ~15-fold, compared with wild-type AAV3 vectors (see e.g. Ling, C., et al., 2014. Human gene therapy, 25(12), pp.1023-1034); a triple mutant vector with S472A, S587A, and N706A substitutions had less reactivity to anti-AAV capsid NAbs than wild-type AAV3 vectors (see e.g. Ito, M., et al., 2021. Scientific Reports, 11 (1 ), p.9322); double mutant AAV-LK03-REDH (R594E + D598H) is a variant LK03 capsid with a superior in vivo transduction in chimeric mice repopulated with primary human hepatocytes (see Cabanes-Creus, M., et al., 2023. Molecular Therapy Methods & Clinical Development, 28, pp.220-237); and a single amino acid insertion at position 265 allows functional transduction of murine hepatocytes with AAV3 capsids (see e.g. Cabanes-Creus, M., et al., 2021. Molecular Therapy Methods & Clinical Development, 21 , pp.607-620).

[0212] AAV3 vector particles which are known in the art include AAV3B vector particles, AAV3A vector particles, AAV-LK03 vector particles, AAV3B-DE5 vector particles, AAV.GT5 vector particles, AAV3-ST vector particles, AAV3B-V04 vector particles, AAV3B-V05 vector particles, AAV3B-G3 vector particles, and AAV3B-E12 vector particle. A sequence alignment between the VP1 capsid sequences of each of these is in shown in Figure 2. Each of the AAV3 VP1 capsid proteins shown in Figure 2 share at least about 95% sequence identity.

[0213] Also, generation of AAV-KP1 , AAV-KP2 and AAV-KP3 vector particles has been described in Pekrun, K., et al., 2019. JCI Insight 2019;4(22):e131610. AAV3B is the parental sequence that is most closely related to AAV-KP1 , AAV-KP2 and AAV-KP3 vector particles; AAV-KP1 and AAV-KP3 demonstrate 92% sequence identity to AAV3B and AAV-KP2 demonstrates 95% identity to AAV3B. The AAV-KP1 capsid sequence contains fragments from at least 7 of the 8 parental serotypes used in the capsid shuffling library whereas AAV-KP2 and AAV-KP3 contained fragments from at least 6 parental serotypes.

[0214] The term ‘AAV3 vector particles’ used herein includes AAV3A vector particles, AAV3B vector particles, AAV3-like vector particles, and variants thereof. That is, ‘AAV3 vector particles’ as used herein comprise an AAV capsid protein having at least 90% identity to the amino acid sequence of SEQ ID NOs: #1# to #10#.

[0215] In some embodiments, the ‘AAV3 vector particles’ are: AAV3B vector particles, AAV3A vector particles, AAV-LK03 vector particles, AAV3B-DE5 vector particles, AAV.GT5 vector particles, AAV3-ST vector particles, AAV3B-V04 vector particles, AAV3B-V05 vector particles, AAV3B-G3 vector particles, AAV3B-E12 vector particles, AAV-KP1 vector particles, AAV-KP2 vector particles, AAV-KP3 vector particles or variants thereof.

[0216] In some embodiments, the AAV3 vector particles are AAV3B or AAV3A vector particles or variants thereof. In some embodiments, the AAV3 vector particles are AAV3B vector particles or variants thereof. In some embodiments, the AAV3 vector particles are AAV3-like vector particles e.g. AAV-LK03 vector particles, AAV3B-DE5 vector particles, AAV.GT5 vector particles, AAV3-ST vector particles, AAV3B-V04 vector particles, AAV3B-V05 vector particles, AAV3B-G3 vector particles, AAV3B-E12 vector particles, AAV-KP1 vector particles, AAV-KP2 vector particles, AAV-KP3 vector particles or variants thereof.

[0217] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of SEQ ID NOs: #1# to #10#. In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: #1# or #2#. In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: #1#.

[0218] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having at least 98.0%, at least 98.1 %, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% identity, or 100.0% identity to any of SEQ ID NOs: #1# to #10#. In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having at least 98.0%, at least 98.1 %, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% identity, or 100.0% identity to SEQ ID NO: #1# or #2#. In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having at least 98.0%, at least 98.1 %, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% identity, or 100.0% identity to SEQ ID NO: #1#. In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 15 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution compared to any of SEQ ID NOs: #1# to #10#. In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 15 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution compared to SEQ ID NO: #1# or #2#. In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 15 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution compared to SEQ ID NO: #1#.

[0219] In some embodiments, the substitutions are selected from the following: K26Q, V29A, Q31 K, R42A, E67A, Q105K, I125L, N735P, Y252F, Y272F, Y444F, F501Y, Y701 F, Y705F, Y731 F, S459V, S663V, T251V, T492V, K528R, K533R, K545E, K545R, Q263A, G450S, T452P, S453G, N457G, Q458T, S459N, R460G, L462K, T492I, A493P, N494S, D495Q, E531 D, E532D, E546Q, T548A, T549G, A550R, S551 D, A553T, L555Y, N557H, S472A, S587A, N706A, R488Q, N583S, R594E, R594G, and D598H. In some embodiments, the substitutions are selected from the following: K26Q, V29A, Q31 K, R42A, E67A, Q105K, I125L, N735P. In some embodiments, the substitutions are selected from the following: Y701 F, Y705F, Y731 F, S459V, S663V, T492V, K528R, K533R, K545E, and K545R. In some embodiments, the substitutions are selected from the following: Q263A, G450S, T452P, S453G, N457G, Q458T, S459N, R460G, L462K, T492I, A493P, N494S, D495Q, E531 D, E532D, K533R, E546Q, T548A, T549G, A550R, S551 D, A553T, L555Y, and N557H. In some embodiments, the substitutions are selected from the following: N557H, R460G, A553T, N494S, S551 D, and L555Y. In some embodiments, the substitutions are selected from the following: S472A, S587A, and N706A. In some embodiments, the substitutions are selected from the following: R488Q, N583S, R594E, R594G, and D598H.

[0220] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having an insertion of 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid compared to any of SEQ ID NOs: #1# to #10#. In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having an insertion of 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid compared to SEQ ID NO: #1# or #2#. In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having an insertion of 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid compared to SEQ ID NO: #1#.

[0221] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having a deletion of 10 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid compared to any of SEQ ID NOs: #1# to #10#. In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having a deletion of 10 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid compared to SEQ ID NO: #1# or #2#. In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having a deletion of 10 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid compared to SEQ ID NO: #1#.

[0222] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having, compared to any of SEQ ID NOs: #1# to #10#: (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) an insertion of 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or les, or 1 amino acid; and / or (c) a deletion of 10 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid.

[0223] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having, compared to SEQ ID NO: #1# or #2#: (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) an insertion of 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or les, or 1 amino acid; and / or (c) a deletion of 10 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid.

[0224] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having, compared to SEQ ID NO: #1#: (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) an insertion of 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or les, or 1 amino acid; and / or (c) a deletion of 10 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid.

[0225] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having, compared to any of SEQ ID NOs: #1# to #10#: (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions,

[0226] 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) no insertions; and (c) no deletions. In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having, compared to SEQ ID NO: #1# or #2#: (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions,

[0227] 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) no insertions; and (c) no deletions. In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having, compared to SEQ ID NO: #1# (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) no insertions; and (c) no deletions.

[0228] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having, compared to any of SEQ ID NOs: #1# to #10#: (a) no substitutions; (b) an insertion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids; and (c) no deletions. In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having, compared to SEQ ID NO: #1# or #2#: (a) no substitutions; (b) an insertion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids; and (c) no deletions. In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having, compared to SEQ ID NO: #1#: (a) no substitutions; (b) an insertion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids; and (c) no deletions.

[0229] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having, compared to any of SEQ ID NOs: #1# to #10#: (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) an insertion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids; and (c) no deletions. In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having, compared to SEQ ID NO: #1# or #2#: (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) an insertion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids; and (c) no deletions. In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having, compared to SEQ ID NO: #1#: (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) an insertion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids; and (c) no deletions.

[0230] AA V3B vector particles

[0231] In some embodiments, the AAV3 vector particles are AAV3B vector particles or variants thereof.

[0232] In some embodiments, the AAV3 vector particles comprise an AAV3B VP1 capsid protein, or variant thereof. In some embodiments, the AAV3 vector particles comprise an AAV3B VP1 capsid protein, an AAV3B VP2 capsid protein, and / or an AAV3B VP3 capsid protein, or variants thereof. In some embodiments, the AAV3 vector particles are encapsidated by AAV3B VP1 , VP2, and VP3 capsid proteins, or variants thereof.

[0233] AAV3B is described in Rutledge, E.A., et al., 1998. Journal of virology, 72(1 ), pp.309- 319. AAV3B VP1 capsid sequence is provided in Uniport entry 056139. An example AAV3B VP1 capsid sequence is provided below in SEQ ID NO: #1#. AAV3B VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: #1#.

[0234] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: #1#.

[0235] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having at least 98.0%, at least 98.1 %, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% identity, or 100.0% identity to the amino acid sequence of SEQ ID NO: #1#.

[0236] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 15 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution compared to SEQ ID NO: #1#.

[0237] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having an insertion of 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid compared to SEQ ID NO: #1#.

[0238] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having a deletion of 10 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid compared to SEQ ID NO: #1#.

[0239] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having, compared to SEQ ID NO: #1#: (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution;

[0240] (b) an insertion of 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or les, or 1 amino acid; and / or

[0241] (c) a deletion of 10 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid.

[0242] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having, compared to SEQ ID NO: #1#: (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) an insertion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids; and (c) no deletions.

[0243] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having, compared to SEQ ID NO: #1#: (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) no insertions; and (c) no deletions.

[0244] AA V3A vector particles

[0245] In some embodiments, the AAV3 vector particles are AAV3A vector particles or variants thereof. In some embodiments, the AAV3 vector particles comprise an AAV3A VP1 capsid protein, or variant thereof. In some embodiments, the AAV3 vector particles comprise an AAV3A VP1 capsid protein, an AAV3A VP2 capsid protein, and / or an AAV3A VP3 capsid protein, or variants thereof. In some embodiments, the AAV3 vector particles are encapsidated by AAV3A VP1 , VP2, and VP3 capsid proteins, or variants thereof.

[0246] AAV3A is described in Muramatsu, S.I., et al., 1996. Virology, 221 (1 ), pp.208-217. An example AAV3A VP1 capsid sequence is provided below in SEQ ID NO: #2#. AAV3A VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: #2#.

[0247] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: #2#.

[0248] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having at least 98.0%, at least 98.1 %, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% identity, or 100.0% identity to the amino acid sequence of SEQ ID NO: #2#.

[0249] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 15 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution compared to SEQ ID NO: #2#.

[0250] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having an insertion of 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid compared to SEQ ID NO: #2#. In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having a deletion of 10 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid compared to SEQ ID NO: #2#.

[0251] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having, compared to SEQ ID NO: #2#:

[0252] (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution;

[0253] (b) an insertion of 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or les, or 1 amino acid; and / or

[0254] (c) a deletion of 10 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid.

[0255] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having, compared to SEQ ID NO: #2#: (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) an insertion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids; and (c) no deletions.

[0256] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having, compared to SEQ ID NO: #2#: (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) no insertions; and (c) no deletions.

[0257] AA V-LK03 vector particles

[0258] In preferred embodiments, the AAV3 vector particles are AAV-LK03 vector particles or variants thereof.

[0259] In some embodiments, the AAV3 vector particles comprise an AAV-LK03 VP1 capsid protein, or variant thereof. In some embodiments, the AAV3 vector particles comprise an AAV-LK03 VP1 capsid protein, an AAV-LK03 VP2 capsid protein, and / or an AAV- LK03 VP3 capsid protein, or variants thereof. In some embodiments, the AAV3 vector particles are encapsidated by AAV-LK03 VP1 , VP2, and VP3 capsid proteins, or variants thereof.

[0260] The AAV-LK03 cap sequence consists of fragments from seven different wild-type serotypes (AAV1 , 2, 3B, 4, 6, 8, 9) and is described in Lisowski, L., et al., 2014. Nature, 506(7488), pp.382-386. An example AAV-LK03 capsid sequence is provided below in SEQ ID NO: #3#. AAV-LK03 VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: #3#.

[0261] AAV-LK03 variants have been described including the insertion of a single amino acid at capsid position 265, i.e. 265insT, and / or a R488Q, N583S, R594E, R594G, D598H substitution (see e.g. Cabanes-Creus, M., et al., 2021. Molecular Therapy Methods & Clinical Development, 21 , pp.607-620; and Cabanes-Creus, M., et al., 2023. Molecular Therapy Methods & Clinical Development, 28, pp.220-237).

[0262] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: #3#.

[0263] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having at least 98.0%, at least 98.1 %, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% identity, or 100.0% identity to the amino acid sequence of SEQ ID NO: #3#.

[0264] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 15 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution compared to SEQ ID NO: #3#.

[0265] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having an insertion of 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid compared to SEQ ID NO: #3#.

[0266] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having a deletion of 10 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid compared to SEQ ID NO: #3#.

[0267] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having, compared to SEQ ID NO: #3#:

[0268] (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution;

[0269] (b) an insertion of 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or les, or 1 amino acid; and / or (c) a deletion of 10 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid.

[0270] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having, compared to SEQ ID NO: #3#: (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) an insertion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids; and (c) no deletions.

[0271] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having, compared to SEQ ID NO: #3#: (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) no insertions; and (c) no deletions.

[0272] In some embodiments, the AAV3 vector particles comprise an AAV capsid protein having the amino acid sequence of SEQ ID NO: #3#.

[0273] AA V3B-DE5 vector particles

[0274] In some embodiments, the AAV3 vector particles are AAV3B-DE5 vector particles or variants thereof.

[0275] In some embodiments, the AAV3 vector particles comprise an AAV3B-DE5 VP1 capsid protein, or variant thereof. In some embodiments, the AAV3 vector particles comprise an AAV3B-DE5 VP1 capsid protein, an AAV3B-DE5 VP2 capsid protein, and / or an AAV3B-DE5 VP3 capsid protein, or variants thereof. In some embodiments, the AAV3 vector particles are encapsidated by AAV3B-DE5 VP1 , VP2, and VP3 capsid proteins, or variants thereof. AAV3B-DE5 contains 24 capsid amino acid substitutions compared with AAV3B and demonstrated improved human hepatocyte tropism in a liver chimeric mouse model (see e.g. Biswas, M., et al., 2020. Molecular Therapy-Methods & Clinical Development, 19, pp.347-361 ). An example AAV3B-DE5 VP1 capsid sequence is provided below in SEQ ID NO: #4#. AAV3B-DE5 VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: #4#.

[0276] AA \ / . GT5 vector particles

[0277] In some embodiments, the AAV3 vector particles are AAV.GT5 vector particles or variants thereof.

[0278] In some embodiments, the AAV3 vector particles comprise an AAV.GT5 VP1 capsid protein, or variant thereof. In some embodiments, the AAV3 vector particles comprise an AAV.GT5VP1 capsid protein, an AAV.GT5VP2 capsid protein, and / or an AAV. GT5 VP3 capsid protein, or variants thereof. In some embodiments, the AAV3 vector particles are encapsidated by AAV.GT5 VP1 , VP2, and VP3 capsid proteins, or variants thereof.

[0279] AAV.GT5 has a high transduction efficiency in human hepatocytes and is relatively insensitive to NAbs found in blood donors (see e.g. Ito, M., et al., 2021. Scientific Reports, 11 (1 ), p.9322). An example AAV.GT5 VP1 capsid sequence is provided below in SEQ ID NO: #5#. AAV.GT5 VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: #5#.

[0280] AAV3-ST vector particles

[0281] In some embodiments, the AAV3 vector particles are AAV3-ST vector particles or variants thereof.

[0282] In some embodiments, the AAV3 vector particles comprise an AAV3-ST VP1 capsid protein, or variant thereof. In some embodiments, the AAV3 vector particles comprise an AAV3-ST VP1 capsid protein, an AAV3-ST VP2 capsid protein, and / or an AAV3- ST VP3 capsid protein, or variants thereof. In some embodiments, the AAV3 vector particles are encapsidated by AAV3-ST VP1 , VP2, and VP3 capsid proteins, or variants thereof. AAV3-ST has a high transduction efficiency for human hepatocytes (see e.g. Vercauteren, K., et al., 2016. Molecular Therapy, 24(6), pp.1042-1049). An example AAV3-ST VP1 capsid sequence is provided below in SEQ ID NO: #6#. AAV3-ST VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: #6#.

[0283] AAV3B-V04 vector particles

[0284] In some embodiments, the AAV3 vector particles are AAV3B-V04 vector particles or variants thereof.

[0285] In some embodiments, the AAV3 vector particles comprise an AAV3B-V04 VP1 capsid protein, or variant thereof. In some embodiments, the AAV3 vector particles comprise an AAV3B-V04 VP1 capsid protein, an AAV3B-V04 VP2 capsid protein, and / or an AAV3B-V04 VP3 capsid protein, or variants thereof. In some embodiments, the AAV3 vector particles are encapsidated by AAV3B-V04 VP1 , VP2, and VP3 capsid proteins, or variants thereof.

[0286] AAV3B-V04 contains 9 amino acid substitutions and provides advantages such as improved human hepatocyte tropism and immune evasion (see e.g. Rana, J., et al., 2023. Human Gene Therapy, 34(7-8), pp.289-302 and WO 2024 / 015877). An example AAV3B-V04 VP1 capsid sequence is provided below in SEQ ID NO: #7#. AAV3B-V04 VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: #7#.

[0287] AAV3B-V05 vector particles

[0288] In some embodiments, the AAV3 vector particles are AAV3B-V05 vector particles or variants thereof.

[0289] In some embodiments, the AAV3 vector particles comprise an AAV3B-V05 VP1 capsid protein, or variant thereof. In some embodiments, the AAV3 vector particles comprise an AAV3B-V05 VP1 capsid protein, an AAV3B-V05 VP2 capsid protein, and / or an AAV3B-V05 VP3 capsid protein, or variants thereof. In some embodiments, the AAV3 vector particles are encapsidated by AAV3B-V05 VP1 , VP2, and VP3 capsid proteins, or variants thereof.

[0290] AAV3B-V05 contains 21 substitutions, relative to wild-type AAV3B (see WO 2024 / 015877). An example AAV3B-V05 VP1 capsid sequence is provided below in SEQ ID NO: #8#. AAV3B-V05 VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: #8#.

[0291] AA V3B-G3 vector particles

[0292] In some embodiments, the AAV3 vector particles are AAV3B-G3 vector particles or variants thereof.

[0293] In some embodiments, the AAV3 vector particles comprise an AAV3B-G3 VP1 capsid protein, or variant thereof. In some embodiments, the AAV3 vector particles comprise an AAV3B-G3 VP1 capsid protein, an AAV3B-G3 VP2 capsid protein, and / or an AAV3B-G3 VP3 capsid protein, or variants thereof. In some embodiments, the AAV3 vector particles are encapsidated by AAV3B-G3 VP1 , VP2, and VP3 capsid proteins, or variants thereof.

[0294] AAV3B-G3 contains 15 amino acid substitutions, relative to wild-type AAV3B (see WO 2021 / 108468). An example AAV3-G3 VP 1 capsid sequence is provided below in SEQ ID NO: #9#. AAV3B-G3 VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: #9#.

[0295] AAV3B-E12 vector particles

[0296] In some embodiments, the AAV3 vector particles are AAV3B-E12 vector particles or variants thereof.

[0297] In some embodiments, the AAV3 vector particles comprise an AAV3B-E12 VP1 capsid protein, or variant thereof. In some embodiments, the AAV3 vector particles comprise an AAV3B-E12 VP1 capsid protein, an AAV3B-E12 VP2 capsid protein, and / or an AAV3B-E12 VP3 capsid protein, or variants thereof. In some embodiments, the AAV3 vector particles are encapsidated by AAV3B-E12 VP1 , VP2, and VP3 capsid proteins, or variants thereof.

[0298] AAV3B-E12 contains 24 amino acid substitutions, relative to wild-type AAV3B (see WO 2021 / 108468). An example AAV3-E 12 VP 1 capsid sequence is provided below in SEQ ID NO: #10#. AAV3B-E12 VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: #10#. AAV vector genomes

[0299] AAV3 vector particles may be transcapsidated forms wherein an AAV genome or derivative having an ITR of any serotype is packaged in an AAV3 capsid or variant thereof.

[0300] An AAV genome may encode functions needed for production of an AAV particle. These functions include those operating in the replication and packaging cycle of AAV in a host cell, including encapsidation of the AAV genome into an AAV particle. Naturally occurring AAVs are replication-deficient and rely on the provision of helper functions in trans for completion of a replication and packaging cycle. Accordingly, an AAV genome is typically replication-deficient.

[0301] An AAV genome may be in single-stranded form (ssAAV), either positive or negativesense, or alternatively in double-stranded form (dsAAV). The use of a double-stranded form allows bypass of the DNA replication step in the target cell and so can accelerate transgene expression. The maximum packaging capacity of the single-stranded form is larger than the double-stranded form. Suitably, an AAV genome is in single-stranded form.

[0302] Typically, an AAV genome comprises at least one inverted terminal repeat sequence (ITR). An ITR sequence acts in cis to provide a functional origin of replication and allows for integration and excision of the vector from the genome of a cell. ITRs may be the only sequences required in cis next to the therapeutic gene.

[0303] An AAV genome may also comprise packaging genes, such as rep and / or cap genes which encode packaging functions for an AAV particle. A promoter may be operably linked to each of the packaging genes. Specific examples of such promoters include the p5, p19 and p40 promoters. For example, the p5 and p19 promoters are generally used to express the rep gene, while the p40 promoter is generally used to express the cap gene. The rep gene encodes one or more of the proteins Rep78, Rep68, Rep52 and Rep40 or variants thereof. The cap gene encodes one or more capsid proteins such as VP1 , VP2 and VP3 or variants thereof.

[0304] An AAV genome may be the full genome of a naturally occurring AAV. Preferably, an AAV genome is derivatised for the purpose of administration to patients. Such derivatisation is standard in the art and the invention encompasses the use of any known derivative of an AAV genome, and derivatives which could be generated by applying techniques known in the art.

[0305] An AAV genome may be a derivative of any naturally occurring AAV. Suitably, an AAV genome is a derivative of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 . Suitably, an AAV genome is a derivative of AAV2.

[0306] Derivatives of an AAV genome include any truncated or modified forms of an AAV genome which allow for expression of a transgene from an AAV vector of the invention in vivo. Typically, it is possible to truncate an AAV genome significantly to include minimal viral sequence yet retain the above function. This is preferred for safety reasons to reduce the risk of recombination of the vector with wild-type virus, and also to avoid triggering a cellular immune response by the presence of viral gene proteins in the target cell.

[0307] Typically, a derivative will include at least one inverted terminal repeat sequence (ITR), preferably more than one ITR, such as two ITRs or more. One or more of the ITRs may be derived from AAV genomes having different serotypes, or may be a chimeric or mutant ITR. A preferred mutant ITR is one having a deletion of a trs (terminal resolution site). This deletion allows for continued replication of the genome to generate a single-stranded genome which contains both coding and complementary sequences, i.e. a self-complementary AAV (scAAV) genome. This allows for bypass of DNA replication in the target cell, and so enables accelerated transgene expression. However, the maximum packaging capacity of a scAAV is reduced.

[0308] An AAV genome may comprise one or more ITR sequences from any naturally derived serotype, isolate or clade of AAV or a variant thereof. An AAV genome may comprise at least one, such as two, AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 ITRs, or variants thereof. Suitably, an AAV genome may comprise at least one, such as two, AAV2 ITRs. Suitably, an AAV genome may comprise two AAV2 ITRs.

[0309] The inclusion of one or more ITRs is preferred to aid concatamer formation of the AAV vector in the nucleus of a host cell, for example following the conversion of singlestranded vector DNA into double-stranded DNA by the action of host cell DNA polymerases. The formation of such episomal concatamers protects the AAV vector during the life of the host cell, thereby allowing for prolonged expression of the transgene in vivo.

[0310] Suitably, ITR elements will be the only sequences retained from the native AAV genome in a derivative. A derivative will preferably not include the rep and / or cap genes of the native genome and any other sequences of the native genome. This is preferred for the reasons described above, and also to reduce the possibility of integration of the vector into the host cell genome. Additionally, reducing the size of the AAV genome allows for increased flexibility in incorporating other sequence elements (such as regulatory elements) within the vector in addition to the proteincoding sequence.

[0311] The following portions could therefore be removed in a derivative: one inverted terminal repeat (ITR) sequence, the replication (rep) and capsid (cap) genes. However, derivatives may additionally include one or more rep and / or cap genes or other viral sequences of an AAV genome. Naturally occurring AAV integrates with a high frequency at a specific site on human chromosome 19, and shows a negligible frequency of random integration, such that retention of an integrative capacity in the AAV vector may be tolerated in a therapeutic setting.

[0312] Protein coding sequences

[0313] The AAV3 vector particles may comprise one or more protein-coding sequences.

[0314] The protein-coding sequence may encode any polypeptide of interest, such as a therapeutic protein. For example, the protein-coding sequence can encode any polypeptide associated with a liver or kidney disease.

[0315] In some embodiments, the protein-coding sequence encodes a polypeptide associated with a kidney disease. In some embodiments, the protein-coding sequence encodes a polypeptide associated with a glomerular disease. In some embodiments, the protein-coding sequence encodes a polypeptide associated with a GBM-associated genetic glomerular disease, such as Alport Syndrome. In some embodiments, the protein-coding sequence encodes a polypeptide involved in podocyte-associated genetic glomerular disease. In some embodiments, the protein-coding sequence encodes a COL4A3, COL4A4, COL4A5, NPHS2, CFH, CFL, FHL-1 , C1 INH, C4BP, MASP2, C3, C5aR1 , C5, C5a, CD55, CD35, CD46, CD59, vitronectin, clusterin, ADCK4, ALG1 , ARHGAP24, ARGHDIA, CD151 , CD2AP, COQ2, COQ6, DGKE, E2F3, EMP2, KANK2, LAGE3, LMNA, LMX1 B, MAF B, NUP85, NUP93, NXF5, OSGEP, PAX2, PDSS2, PMM2, PODXL, SCARB2, SGPL1 , Smad7, TP53RK, TPRKB, VDR, WDR73, WT1 , ZMPSTE24, APOL1 , NPHS1 , TRPC6, NUP107, NUP133, NUP160, ACTN4, INF2, ANKFY1 , ANLN, CRB2, ITGA3, KANK1 , KANK4, MAGI2, MY01 E, OCRL, PTPRO, SMARCAL1 , SYNPO, TBC1 D8B, XPO5, TNS2, NLRP3, or VEGFC polypeptide.

[0316] In some embodiments, the protein-coding sequence encodes a NPHS2, COL4A3, COL4A4 or COL4A5, CFI, CFH, FHL-1 , and / or VEGFC polypeptide. In some embodiments, the protein-coding sequence encodes a NPHS2 polypeptide. In some embodiments, the protein-coding sequence encodes a COL4A3, COL4A4 or COL4A5 polypeptide. In some embodiments, the protein-coding sequence encodes a CFI, CFH, and / or FHL-1 polypeptide. In some embodiments, the protein-coding sequence encodes a VEGFC polypeptide.

[0317] For a protein-coding polynucleotide, it will be understood by a skilled person that numerous different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the invention to reflect the codon usage of any particular host organism in which the polypeptides of the invention are to be expressed.

[0318] The protein-coding sequence may be codon-optimised. Different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type. By altering the codons in the sequence so that they are tailored to match with the relative abundance of corresponding tRNAs, it is possible to increase expression. By the same token, it is possible to decrease expression by deliberately choosing codons for which the corresponding tRNAs are known to be rare in the particular cell type. Thus, an additional degree of translational control is available. Codon usage tables are known in the art for mammalian cells (e.g. humans), as well as for a variety of other organisms. NPHS2

[0319] The one or more protein-coding sequences may encode NPHS2, or a fragment and / or variant thereof.

[0320] “NPHS2” is the abbreviated name of the polypeptide encoded by the NPHS2 gene and is also known as podocin. NPHS2 is a 42kDa hairpin like membrane-associated podocyte-specific protein that is a key component of the protein complex at the slit diaphragm; the cell-cell junction between adjacent podocyte foot processes. It localises to lipid rafts and interacts with other important slit diaphragm proteins like nephrin, CD2AP and TRPC6. It is essential in the maintenance of the slit diaphragm, and consequently the integrity of the glomerular filtration barrier.

[0321] A fragment and / or variant of NPHS2 may retain NPHS2 activity and / or function. For example, a fragment and / or variant of podocin may regulate glomerular permeability. Suitably, a fragment and / or variant of NPHS2 may have the same or similar activity and / or function to NPHS2, e.g. may have at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the activity and / or function of NPHS2.

[0322] A person skilled in the art would be able to generate fragments and / or variants using conservative substitutions, based on the known structural and functional features of NPHS2 (see e.g. Tabassum, A., et al., 2014. Interdisciplinary Sciences: Computational Life Sciences, 6(1 ), pp.32-39), and / or based on known variants (see e.g. NCBI Gene ID: 7827 and NCBI HomoloGene: 22826). Suitably, a fragment and / or variant of NPHS2 comprises a transmembrane domain, with two cytoplasmic domains at the N- and C-terminus.

[0323] The NPHS2 gene is conserved in chimpanzee, Rhesus monkey, dog, cow, mouse, and rat. The NPHS2 may be a human NPHS2. Suitably, the NPHS2 may comprise or consist of a polypeptide sequence of UniProtKB accession Q9NP85, or a fragment and / or variant thereof.

[0324] In some embodiments, the NPHS2 comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: #11# or a fragment thereof. Suitably, the NPHS2 comprises or consists of an amino acid sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: #11# or a fragment thereof.

[0325] In some embodiments, the NPHS2 comprises or consists of the amino acid sequence of SEQ ID NO: #11# or a fragment thereof.

[0326] COL4A3, COL4A4 and COL4A5 polypeptides

[0327] The protein-coding sequence may encode a COL4A3, COL4A4 or COL4A5 polypeptide, or a fragment and / or variant thereof.

[0328] COL4A3, COL4A4 and COL4A5 proteins are approximately 170-185 kDa homologous polypeptides containing collagenous Gly-X-Y repeat sequences frequently interrupted by non-collagenous sequences and forming a triple helix repeat. Each polypeptide also contains a large globular non-collagenous domain at the carboxyl-terminal end.

[0329] Alport syndrome (AS) is caused by pathogenic variants in the COL4A3, COL4A4 and COL4A5 genes, which result in abnormalities of the collagen IV a345 network of basement membranes. The COL4A3, COL4A4 or COL4A5 polypeptide or a fragment or derivative thereof may be capable of forming a collagen IV a345 network.

[0330] Approximately 200-300 amino acids may be removed from each of the COL4A3, COL4A4 and COL4A5 polypeptides to produce a truncated transgene suitable for a mini-gene approach. The amino acids may be removed from the triple helix repeat. Preferably the amino acids are not removed from the non-collagenous region.

[0331] In some embodiments the COL4A3, COL4A4 and COL4A5 polypeptides are full-length polypeptides.

[0332] Preferably, the COL4A3, COL4A4 or COL4A5 polypeptide is human. An example human COL4A3 is the COL4A3 having the UniProtKB accession number Q01955. An example human COL4A4 is the COL4A3 having the UniProtKB accession number P53420. An example human COL4A5 is the COL4A5 having the UniProtKB accession number P29400.

[0333] Suitably, the COL4A3 peptide may comprise or consist of the polypeptide sequence shown as SEQ ID NO: #12#, or a variant which is at least 70% identical to SEQ ID NO: #12#. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: #12#.

[0334] Suitably, the COL4A4 peptide may comprise or consist of the polypeptide sequence shown as SEQ ID NO: #13#, or a variant which is at least 70% identical to SEQ ID NO: #13#. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: #13#.

[0335] Suitably, the COL4A5 peptide may comprise or consist of the polypeptide sequence shown as SEQ ID NO: #14#, or a variant which is at least 70% identical to SEQ ID NO: #14#. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: #14#.

[0336] Complement Factor I (CFI)

[0337] The one or more protein-coding sequences may encode CFI, or a fragment and / or variant thereof.

[0338] Complement factor I (CFI) is a trypsin-like serine protease that inhibits the complement system by cleaving three peptide bonds in the alpha-chain of C3b and two bonds in the alpha-chain of C4b thereby inactivating these proteins.

[0339] CFI is a glycoprotein heterodimer consisting of a disulfide linked heavy chain and light chain. The heavy chain has four domains: an Fl membrane attack complex (FIMAC) domain, CD5 domain, and low density lipoprotein receptor 1 and 2 (LDLrl and LDLr2) domains. The heavy chain plays an inhibitory role in maintaining the enzyme inactive until it meets the complex formed by the substrate (either C3b or C4b) and a cofactor protein (Factor H, C4b-binding protein, complement receptor 1 , and membrane cofactor protein). Upon binding of the enzyme to the substrate: cofactor complex, the heavy: light chain interface is disrupted, and the enzyme activated by allostery. The light chain contains only the serine protease domain. This domain contains the catalytic triad His-362, Asp-411 , and Ser-507, which is responsible for specific cleavage of C3b and C4b. The CFI or a fragment and / or variant thereof may be capable of cleaving C3b into iC3b and / or may be capable of cleaving iC3b into C3d,g. The fragment and / or variant of CFI may retain at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of the C3b- inactivating and iC3b-degradation activity of native CFI. The C3b-inactivating and iC3b-degradation activity of the fragment and / or variant of CFI and native CFI, may be determined using any suitable method known to those of skill in the art. For example, using a proteolytic assay.

[0340] Preferably, the CFI is a human CFI. An example human CFI is the CFI having the UniProtKB accession number P05156.

[0341] In some embodiments, the CFI comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: #15# or a fragment thereof. Suitably, the CFI comprises or consists of an amino acid sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: #15# or a fragment thereof.

[0342] In some embodiments, the CFI comprises or consists of the amino acid sequence of SEQ ID NO: #15# or a fragment thereof.

[0343] Complement Factor H (CFH)

[0344] The one or more protein-coding sequences may encode CFH, or a fragment and / or variant thereof.

[0345] Complement factor H (CFH) regulates complement activation on self cells and surfaces. CFH competes for binding of complement factor B (CFB) to C3b, acts as a cofactor for CFI-catalysed proteolytic cleavage of C3b, and accelerates the irreversible dissociation of C3bBb and C3b2Bb into their separate components. Thus, CFH not only inhibits formation of the convertases but it also shortens the lifespan of any convertase complex that forms.

[0346] CFH is a large (155 kDa) soluble glycoprotein. CFH is composed from a total of 20 domains, each containing approximately 60 amino acid residues and termed complement control protein modules (CCPs) or short consensus repeats that are joined by short linkers consisting of 3-8 residues. The CCP modules are numbered from 1-20 (from the N-terminus of the protein): CCPs 1-4 and CCPs 19-20 engage with C3b while CCPs 7 and CCPs 19-20 bind to GAGs and sialic acid.

[0347] The CFH or a fragment and / or variant thereof may be capable of binding C3b and / or C3d; and / or acting as a cofactor for the CFI-catalysed proteolytic cleavage of C3b; and / or increasing the irreversible dissociation of C3bBb and C3b2Bb into their separate components. The fragment and / or variant of CFH may retain at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of the activity of native CFH. The activity of the fragment and / or variant of CFH and native CFH may be determined using any suitable method known to those of skill in the art.

[0348] Preferably, the CFH is a human CFH. An example human CFH is the CFH having the UniProtKB accession number P08603.

[0349] In some embodiments, the CFH comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: #16# or a fragment thereof. Suitably, the CFH comprises or consists of an amino acid sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: #16# or a fragment thereof.

[0350] In some embodiments, the CFH comprises or consists of the amino acid sequence of SEQ ID NO: #16# or a fragment thereof.

[0351] Factor H-like protein 1 (FHL-1)

[0352] The one or more protein-coding sequences may encode FHL-1 , or a fragment and / or variant thereof.

[0353] FHL-1 or a fragment and / or variant thereof may be capable of binding C3b and / or C3d. The fragment and / or variant of FHL-1 may retain at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of the activity of native FHL-1 . The activity of the fragment and / or variant of FHL-1 and native FHL-1 may be determined using any suitable method known to those of skill in the art.

[0354] Preferably, the FHL-1 is a human FHL-1. An example human FHL-1 is the FHL-1 having the NCBI Reference Sequence: NP_001014975.1 . In some embodiments, the FHL-1 comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: #17# or a fragment thereof. Suitably, the FHL-1 comprises or consists of an amino acid sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: #17# or a fragment thereof.

[0355] In some embodiments, the FHL-1 comprises or consists of the amino acid sequence of SEQ ID NO: #17# or a fragment thereof.

[0356] Vascular endothelial growth factor (VEGF)C polypeptides

[0357] The protein-coding sequence may encode a vascular endothelial growth factor (VEGF)C polypeptide, or a fragment and / or variant thereof.

[0358] VEGFC is a lymphangiogenic growth factor, which is known to signal via two receptors, VEGFR-3 (Flt-4) and VEGFR-2 (Flk-4). VEGFC is produced by cells in a prepropeptide form, which dimerises before being cleaved into a tetramer.

[0359] The protein-coding sequence may encode any form of VEGFC, such as the prepropeptide form, the tetramer form, the intermediate form, or fully processed mature VEGFC.

[0360] If desired, protein-coding sequences encoding different forms of VEGFC polypeptides may be used in any combination. Preferably, the protein-coding sequence comprises a polynucleotide encoding one or more polypeptides having VEGFC biological activity, i.e., peptides that can bind to and activate VEGFR-2 and / or VEGRF-3. More preferably, the protein-coding sequence comprises a polynucleotide encoding a polypeptide comprising the VEGFC homology domain and having VEGFC biological activity, i.e., a polypeptide that can bind to and activate VEGFR-2 and / or VEGRF-3. Further details of suitable VEGFC polynucleotides and polypeptides include those described in WO 2015 / 022447 and US 2014 / 0087002.

[0361] The variant sequence may encode a VEGFC polypeptide that has retained the capability to bind and activate VEGFR-2 and VEGFR-3.

[0362] In some embodiments, the VEGFC polypeptide comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: #18# or a fragment thereof. Suitably, the VEGFC polypeptide comprises or consists of an amino acid sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: #18# or a fragment thereof.

[0363] In some embodiments, the VEGFC polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: #18# or a fragment thereof.

[0364] Regulatory elements

[0365] The AAV3 vector particles may comprise one or more regulatory sequences which may act pre- or post-transcriptionally. Suitably, a protein-coding sequence may be operably linked to one or more regulatory sequences. The one or more regulatory sequences may facilitate expression of the protein.

[0366] “Regulatory sequences” are any sequences which facilitate expression of the polypeptides, e.g. act to increase expression of a transcript or to enhance mRNA stability. Suitable regulatory sequences include for example promoters, enhancer elements, post-transcriptional regulatory elements and polyadenylation sites.

[0367] Promoters

[0368] The AAV3 vector particles may comprise a promoter. Suitably, the promoter may be operably linked to a protein-coding sequence. The promoter may facilitate expression of the protein.

[0369] A “promoter” is a region of DNA that leads to initiation of transcription of a gene. Promoters are located near the transcription start sites of genes, upstream on the DNA (towards the 5' region of the sense strand). Any suitable promoter may be used, the selection of which may be readily made by the skilled person.

[0370] Suitably, the promoter is operable in mammalian cells, e.g. human cells. The promoter may be capable of driving expression of a protein-coding sequence in mammalian cells, e.g. human cells. The promoter may be a mammalian promoter, e.g. a human promoter. The promoter may be operable in liver cells and / or kidney cells. In some embodiments, the promoter is operable in kidney cells. The promoter may be capable of driving expression of a protein-coding sequence in the kidney. Examples of kidney cells include, but are not limited to glomerular cells. In some embodiments, the promoter is operable in glomerular cells. The promoter may be capable of driving expression of a protein-coding sequence in the glomerulus. In some embodiments, the promoter is operable in a podocyte cell. The promoter may be capable of driving expression of a protein-coding sequence in podocytes.

[0371] The promoter may be a minimal promoter. As used, herein, a “minimal promoter” means the minimal sequence that can act as a promoter.

[0372] The promoter may be a constitutive promoter, an inducible promoter, or a repressible promoter. The promoter may be a ubiquitous promoter or a tissue-specific promoter.

[0373] In some embodiments, the promoter is a constitutive promoter. As used herein, a “constitutive promoter” is a promoter which is always active. Suitable constitutive promoters will be known to the skilled person. Example constitutive promoters include the CMV promoter, the EF1 a promoter, the CAG promoter, the PGK promoter, the U6 promoter, the T7 promoter, the SV40 promoter, and the Sp6 promoter.

[0374] In some embodiments, the promoter is an inducible promoter. An “inducible promoter” may refer to a promoter which is activated in response to specific stimuli (e.g. in response to chemicals, temperature, or light). Suitable inducible promoters will be known to the skilled person. In some embodiments, the promoter is a repressible promoter. A “repressible promoter” may refer to a promoter which is de-activated in response to specific stimuli. Suitable repressible promoters will be known to the skilled person.

[0375] In some embodiments, the promoter is a ubiquitous promoter. As used herein, a “ubiquitous promoter” is a promoter which is active in a wide range of cells and tissues.

[0376] In other embodiments, the promoter is a cell-specific or tissue-specific promoter. As used herein, a “cell-specific promoter” or “tissue-specific promoter” is a promoter which preferentially facilitates expression of a protein-coding sequence in a specific type of cells or tissue (see e.g. Zheng, C. and Baum, B.J., 2008. Gene Therapy Protocols: Design and Characterization of Gene Transfer Vectors, pp.205-219). Suitably, a cellspecific or tissue-specific promoter may facilitate higher expression of a protein-coding sequence in one cell-type or tissue as compared to other cell-types or tissues. For example, a cell-specific or tissue-specific promoter may be a promoter which facilitates expression of a protein-coding sequence at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 100% higher, at least 200% higher, at least 300% higher, at least 400% higher, at least 500% higher, or at least 1000% higher in one cell-type or tissue as compared to expression in other cell-types or tissues.

[0377] Suitable tissue-specific promoters will be known to the skilled person (see e.g. Toscano, M.G., et al., 2011 . Gene therapy, 18(2), pp.117-127; and Powell, S.K., et al., 2015. Discovery medicine, 19(102), p.49) and can be generated using methods known in the art (see and Shen, S.Q., et al., 2016. Genome research, 26(2), pp.238-255). In some embodiments, the promoter is a kidney-specific promoter, a neuron-specific promoter, an astrocyte-specific promoter, an oligodendrocyte-specific promoter, a retina-specific promoter, a lung-specific promoter, a liver-specific promoter, a pancreas-specific promoter, a cardiac-specific promoter, or a skeletal muscle-specific promoter.

[0378] In some embodiments, the promoter is a liver-specific promoter or a kidney-specific promoter.

[0379] In some embodiments, the promoter is a kidney-specific promoter. Suitable kidneyspecific promoters will be known to the skilled person. In some embodiments, the promoter is a glomerular-specific promoter. Suitable glomerular-specific promoters will be known to the skilled person. In some embodiments, the promoter is a podocytespecific promoter. Suitable podocyte-specific promoters will be known to the skilled person.

[0380] In some embodiments, the promoter is a NPHS1 or a NPHS2 promoter, or a variant thereof.

[0381] In some embodiments, the promoter is a minimal kidney-specific promoter. As used, herein, a “minimal kidney-specific promoter” means the minimal sequence that can act as a kidney-specific promoter. In some embodiments, the promoter is a minimal glomerular-specific promoter. In some embodiments, the promoter is a minimal podocyte-specific promoter.

[0382] In some embodiments, the promoter is a minimal NPHS1 or a minimal NPHS2 promoter, or a variant thereof.

[0383] In some embodiments, the promoter is a human promoter, e.g. a minimal human NPHS1 promoter or a minimal human NPHS2 promoter.

[0384] NPHS1 promoter

[0385] In some embodiments, the promoter is a NPHS1 promoter, or a variant thereof.

[0386] The NPHS1 gene encodes nephrin, which is selectively expressed in podocytes. A human NPHS1 promoter has been described in Moeller et al. 2002 J Am Soc Nephrol, 13(6): 1561 -7 and Wong MA et al. 2000 Am J Physiol Renal Physiol, 279(6): F1027- 32. This NPHS1 promoter is a 1.2kb fragment and appears to be podocyte-specific. The 1 ,2kb promoter region lacks a TATA box, but has recognition motifs for other transcription factors e.g. PAX-2 binding element, E-box and GATA consensus sequences.

[0387] Suitably, the NPHS1 promoter may comprise or consist of the nucleotide sequence shown as SEQ ID NO: #19#, or a variant which is at least 70% identical to SEQ ID NO: #19#. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: #19#.

[0388] The NPHS1 promoter may be a minimal NPHS1 promoter. Suitably, the NPHS1 promoter has a length of about 1.1 kb or less, about 1.0 kb or less, about 0.9 kb or less, about 0.8 kb or less, about 0.7 kb or less, about 0.6 kb or less, about 0.5 kb or less, about 0.4 kb or less, or about 0.3 kb or less. Suitably, the NPHS1 promoter has a length of about 265 bp or more. In some embodiments, the NPHS1 promoter has a length of about 265-1100 bp, 265-1000 bp, 265-900 bp, 265-800 bp, 265-700 bp, 265- 600 bp, 265-500 bp, 265-400 bp, or 265-300 bp.

[0389] In some embodiments, the NPHS1 promoter may comprise or consist of the nucleotide sequence shown as SEQ ID NO: #20#, or a variant which is at least 70% identical to SEQ ID NO: #20#. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: #20#.

[0390] NPHS2 promoter

[0391] In some embodiments, the promoter is a NPHS2 promoter, or a variant thereof.

[0392] The NPHS2 gene encodes podocin, which is selectively expressed in podocytes. A human NPHS2 promoter has been described in Oleggini R, et al., 2006. Gene Expr. 13(1 ):59-66.

[0393] Suitably, the NPHS2 promoter may comprise or consist of the nucleotide sequence shown as SEQ ID NO: #21#, or a variant which is at least 70% identical to SEQ ID NO: #21#. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: #21#.

[0394] The NPHS2 promoter may be a minimal NPHS2 promoter. Suitably, the NPHS2 promoter has a length of about 2.0 kb or less, about 1.8 kb or less, about 1.6 kb or less, about 1.4 kb or less, about 1.2 kb or less, about 1.0 kb or less, about 0.9 kb or less, about 0.8 kb or less, or about 0.7 kb or less. Suitably, the NPHS2 promoter has a length of about 628 bp or more. In some embodiments, the NPHS2 promoter has a length of about 628-2000 bp, 628-1800 bp, 628-1600 bp, 628-1400 bp, 628-1200 bp, 628-1000 bp, 628-900 bp, 628-800 bp, or 628-700 bp.

[0395] In some embodiments, the NPHS2 promoter may comprise or consist of the nucleotide sequence shown as SEQ ID NO: #22#, or a variant which is at least 70% identical to SEQ ID NO: #22#. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: #22#.

[0396] Hybrid promoters

[0397] In some embodiments, the promoter is a hybrid enhancer-promoter. In some embodiments, the hybrid enhancer-promoter is a hybrid kidney-specific promoter comprising a kidney-specific enhancer and a ubiquitous or core promoter. In some embodiments, the hybrid enhancer-promoter is a hybrid glomerular-specific promoter comprises a glomerular-specific enhancer and a ubiquitous or core promoter. In some embodiments, the hybrid enhancer-promoter is a hybrid podocytespecific promoter comprises a podocyte-specific enhancer and a ubiquitous or core promoter.

[0398] Suitably, the enhancer may be or may be derived from an enhancer associated with a gene with selective expression in human podocytes. Methods to identify the enhancer regions associated with genes will be well known to those of skill in the art. Preferably, the enhancer is a NPHS1 or a NPHS2 enhancer, or a fragment and / or variant thereof. Preferably, the enhancer is a human enhancer, e.g. a human NPHS1 enhancer or human NPHS2 enhancer.

[0399] A NPHS1 enhancer has been described in Guo, G., et al., 2004. Journal of the American Society of Nephrology, 15(11 ), pp.2851 -2856. A 186-bp fragment from the human NPHS1 promoter was capable of directing podocyte-specific expression of a [3-galactosidase transgene when placed in front of a heterologous minimal promoter in transgenic mice.

[0400] Suitably, a NPHS1 enhancer may comprise or consist of the nucleotide sequence shown as SEQ ID NO: #23#, or a variant which is at least 70% identical to SEQ ID NO: #23#. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: #23#.

[0401] A NPHS2 enhancer has been described in WO 2023 / 213738. The NPHS2 enhancer comprises NPHS2 motif Lmx1 b-FoxC2 and allowed the selective expression of genes of interest in podocytes and other kidney cell lines.

[0402] Suitably, a NPHS2 enhancer may comprise or consist of the nucleotide sequence shown as SEQ ID NO: #24#, or a variant which is at least 70% identical to SEQ ID NO: #24#. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: #24#. The enhancer may be upstream of a ubiquitous promoter or core promoter. The enhancer and the ubiquitous promoter or core promoter may be operably linked. In some embodiments, the enhancer is upstream of a core promoter. In some embodiments, the enhancer is upstream of a super core promoter.

[0403] The “core promoter” is typically 80 nucleotides long, encompassing from -40 to +40 relative to the transcription start site and consists of several functional sub-regions, termed core elements or motifs.

[0404] A “super core promoter” may refer to a synthetic core promoter which contains a combination of core promoter elements that drive high levels of transcription. For example, a super core promoter may contain a TATA box, an initiator motif (Inr), a motif ten element (MTE) and a downstream promoter element (DPE). Example super core promoters have been described in Juven-Gershon, T., et al., 2006. Nature methods, 3(11 ), pp.917-922 and Even, D.Y., et al., 2016. PloS one, 11 (2), p.e0148918.

[0405] A super core promoter may comprise from 5’ to 3’: a TATA box, an initiator motif (Inr), a motif ten element (MTE), and a downstream promoter element (DPE). The super core promoter may comprise one or more further core promoter elements, such as transcription binding sites (e.g. TFIIB recognition element).

[0406] Suitably, the super core promoter is selected from super core promoter 1 (SCP1 ), super core promoter 2 (SCP2), or super core promoter 3 (SCP3). In some embodiments, the super core promoter is SCP1 .

[0407] In some embodiments, the super core promoter comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: #25#. Suitably, the super core promoter comprises or consists of a nucleotide sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: #25#. In some embodiments, the super core promoter comprises or consists of the nucleotide sequence SEQ ID NO: #25#.

[0408] In some embodiments, the hybrid enhancer-promoter comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: #26#. Suitably, the hybrid enhancer-promoter comprises or consists of a nucleotide sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: #26#. In some embodiments, the hybrid enhancerpromoter comprises or consists of the nucleotide sequence SEQ ID NO: #26#.

[0409] Enhancers

[0410] The AAV3 vector particles may comprise an enhancer. Suitably, the enhancer may be operably linked to a protein-coding sequence. The enhancer may facilitate expression of the protein.

[0411] An “enhancer” is a region of DNAthat can be bound by proteins (activators) to increase the likelihood that transcription of a particular gene will occur. Enhancers are cisacting. They can be located up to 1 Mbp (1 ,000,000 bp) away from the gene, upstream or downstream from the start site. Any suitable enhancer may be used, the selection of which may be readily made by the skilled person.

[0412] In some embodiments, the enhancer is a NPHS1 or a NPHS2 enhancer, or a fragment or derivative thereof. In some embodiments, the enhancer is a human enhancer, e.g. a human NPHS1 enhancer or human NPHS2 enhancer.

[0413] The enhancer may be used with the corresponding promoter, for example the NPHS1 enhancer may be used with the NPHS1 promoter. Alternatively, the enhancer may be used with a different promoter.

[0414] Kozak sequence

[0415] The AAV3 vector particles may comprise a Kozak sequence. Suitably, the Kozak sequence may be operably linked to a protein-coding sequence. A Kozak sequence may be inserted before the start codon of the protein to improve the initiation of translation.

[0416] Suitable Kozak sequences will be well known to those of skill in the art (see e.g. Kozak, M., 2002. Gene, 299(1-2), pp.1 -34). A consensus Kozak sequence in vertebrates may have the sequence of SEQ ID NO: #27# or SEQ ID NO: #28#. Suitably, the Kozak sequence may comprise or consist of the nucleotide sequence of SEQ ID NO: #27# or #28#, or variants thereof which have five or fewer deletions, substitutions or insertions. Suitably, the variants may have four or fewer, three or fewer, two or fewer, or one deletion(s), substitution(s) or insertion(s). Suitably, the variants may have three or fewer, two or fewer, or one deletion(s) and / or three or fewer, two or fewer, or one substitution(s). Suitably, the variants may have three or fewer, two or fewer, or one deletion(s) and / or three or fewer, two or fewer, or one substitution(s). Suitably, the variants may have one deletion and / or one substitution. Suitably, the variants may have one deletion and one substitution.

[0417] Post-transcriptional regulatory elements

[0418] The AAV3 vector particles may comprise a post-transcriptional regulatory element. Suitably, the post-transcriptional regulatory element may be operably linked to a protein-coding sequence. Suitably, the transcriptional regulatory element may be downstream of a protein-coding sequence.

[0419] The AAV3 vector particles may comprise a Woodchuck Hepatitis Virus Post- transcriptional Regulatory Element (WPRE). Suitably, the WPRE may be operably linked to the protein-coding sequence. Suitably, the transcriptional regulatory element may be downstream of the protein-coding sequence.

[0420] The WPRE sequence may have mutations within the X-antigen promoter and / or the initiation codon of the X-antigen. This may prevent the production of a functional X- antigen. Suitably, the WPRE may comprise or consist of the nucleotide sequence shown as SEQ ID NO: #29#, or a variant which is at least 70% identical to SEQ ID NO: #29#. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: #29#.

[0421] Polyadenylation sequence

[0422] The AAV3 vector particles may comprise a polyadenylation sequence. Suitably, the polyadenylation sequence may be operably linked to a protein-coding sequence. Suitably, the polyadenylation sequence may be downstream of a protein-coding sequence. A “polyadenylation sequence” is a region of DNA or mRNA that comprises the elements required for polyadenylation of the mRNA. The sequence elements for polyadenylation typically include a polyadenylation signal (with consensus sequence AATAAA) and a polyadenylation site (consensus sequence CA) and may also include a GT-rich downstream element.

[0423] Suitable polyadenylation sequences are well-known in the art and may include a bovine growth hormone polyadenylation sequence (bGH), a soluble neuropilin-1 polyadenylation sequence, an early SV40 polyadenylation sequence (SV40pA), and a chicken beta-globin polyadenylation sequence.

[0424] Suitably, the polyadenylation sequence may comprise or consist of the nucleotide sequence shown as SEQ ID NO: #30#, or a variant which is at least 70% identical to SEQ ID NO: #30#. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: #30#.

[0425] Suitably, the polyadenylation sequence may comprise or consist of the nucleotide sequence shown as SEQ ID NO: #31#, or a variant which is at least 70% identical to SEQ ID NO: #31#. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: #31#.

[0426] Suitably, the polyadenylation sequence may comprise or consist of the nucleotide sequence shown as SEQ ID NO: #32#, or a variant which is at least 70% identical to SEQ ID NO: #32#. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: #32#.

[0427] Suitably, the polyadenylation sequence may comprise or consist of the nucleotide sequence shown as SEQ ID NO: #33#, or a variant which is at least 70% identical to SEQ ID NO: #33#. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: #33#.

[0428] Spacer sequences

[0429] The AAV3 vector particles may further comprise one or more further nucleotide sequences, for example one or more spacer sequence.

[0430] As used herein, a “spacer sequence” may refer to any nucleotide sequence which is inserted between two elements, such that the elements are in a relationship permitting them to function in their intended manner. Suitably, spacer sequences may be included to provide a polynucleotide of a desired length. For example, for efficient encapsidation of AAV vectors, spacer sequences may be included if the expression cassette is less than 3.0 kb in length. Suitably, a spacer sequence has no function or activity.

[0431] The length and sequence of a spacer sequence is not particularly limited. Suitably, a spacer sequence has a length of about 1000 bp or less, about 900 bp or less, about 800 bp or less, about 700 bp or less, about 600 bp or less, about 500 bp or less, about 400 bp or less, about 300 bp or less, about 200 bp or less, about 180 bp or less, about 160 bp or less, about 140 bp or less, about 120 bp or less, or about 100 bp or less. Suitably, a spacer sequence has a length of about 1 bp or more, about 2 bp or more, about 3 bp or more, about 4 bp or more, about 5 bp or more, or about 10 bp or more. Suitably, a spacer sequence has a length of about 1 bp to about 1000 bp, about 1 bp to about 500 bp, about 1 bp to about 200 bp, or about 10 bp to about 100 bp. Suitably, a spacer sequence has an arbitrary sequence that lacks function or activity (e.g. lacks binding sites) (see e.g. Estrada, J., et al., 2016. PloS one, 11 (3), p.e0151740).

[0432] Variants, derivatives, homologues and fragments

[0433] In addition to the specific proteins and nucleotides mentioned herein, the invention also encompasses variants, derivatives, homologues and fragments thereof.

[0434] In the context of the invention, a “variant” of any given sequence may refer a sequence in which the specific sequence of residues (whether amino acid or nucleic acid residues) has been modified in such a manner that the polypeptide or polynucleotide in question retains at least one or all of its endogenous functions. A variant sequence can be obtained by addition, deletion, substitution, modification, replacement and / or variation of at least one residue present in the given sequence.

[0435] The term “derivative” as used herein in relation to proteins or polypeptides of the invention includes any substitution of, variation of, modification of, replacement of, deletion of and / or addition of one (or more) amino acid residues from or to the sequence, providing that the resultant protein or polypeptide retains at least one or all of its endogenous functions.

[0436] Typically, amino acid substitutions may be made, for example from 1 , 2 or 3, to 10 or 20 substitutions, provided that the modified sequence retains the required activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogues.

[0437] Polypeptides used in the invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or the amphipathic nature of the residues as long as the endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine.

[0438] Conservative substitutions may be made, for example according to the table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

[0439] The effect of additions, deletions, substitutions, modifications, replacements and / or variations may be predicted using any suitable prediction tool e.g. SIFT (Vaser, R., et al., 2016. Nature protocols, 11 (1 ), pp.1 -9), PolyPhen-2 (Adzhubei, I., et al., 2013. Current protocols in human genetics, 76(1 ), pp.7-20), CADD (Rentzsch, P., et al., 2021. Genome medicine, 13(1 ), pp.1 -12), REVEL (loannidis, N.M., et al., 2016. The American Journal of Human Genetics, 99(4), pp.877-885), MetaLR (Dong, C., et al., 2015. Human molecular genetics, 24(8), pp.2125-2137), and / or MutationAssessor (Reva, B., et al., 2011. Nucleic acids research, 39(17), pp.el 18-e118) or based on clinical data e.g. ClinVar (Landrum, M.J., et al., 2016. Nucleic acids research, 44(D1 ), pp.D862-D868). Suitable additions, deletions, substitutions, modifications, replacements and / or variations may be considered tolerated, benign, and / or likely benign.

[0440] In the present context, a variant sequence is taken to include an amino acid sequence which may be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical, suitably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the subject sequence. Although a variant can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties / functions), in the context of the present invention it is preferred to express it in terms of sequence identity.

[0441] In the present context, a variant sequence is taken to include a nucleotide sequence which may be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical, suitably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the subject sequence. Although a variant can also be considered in terms of similarity, in the context of the present invention it is preferred to express it in terms of sequence identity.

[0442] The term “homologue” as used herein means a variant having a certain similarity with the wild type amino acid sequence or the wild type nucleotide sequence, e.g. having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% similarity, suitably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similarity to the subject sequence.

[0443] Suitably, reference to a sequence which has a percent identity to any one of the SEQ ID NOs detailed herein refers to a sequence which has the stated percent identity over the entire length of the SEQ ID NO referred to.

[0444] Sequence identity comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percent identity between two or more sequences.

[0445] Percent identity may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

[0446] Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion in the amino acid or nucleotide sequence may cause the following residues or codons to be put out of alignment, thus potentially resulting in a large reduction in percent identity when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall identity score. This is achieved by inserting “gaps” in the sequence alignment to try to maximise local identity.

[0447] However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids or nucleotides, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. “Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0448] Calculation of maximum percent identity therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (see e.g. Devereux, J., et al., 1984. Nucleic acids research, 12(1 ), pp.387-395). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see e.g. Altschul, S.F., et al., 1990. Journal of molecular biology, 215(3), pp.403-410), BLAST 2 (see e.g. Tatusova, T.A. and Madden, T.L., 1999. FEMS microbiology letters, 174(2), pp.247-250), FASTA (see e.g. Pearson, W.R. and Lipman, D.J., 1988. PNAS, 85(8), pp.2444-2448.), EMBOSS Needle (Madeira, F., et al., 2019. Nucleic acids research, 47(W1 ), pp.W636-W641 ) and the GENEWORKS suite of comparison tools. For some applications, it is preferred to use EMBOSS Needle.

[0449] Although the final percent identity can be measured, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix.

[0450] Once the software has produced an optimal alignment, it is possible to calculate percent sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. The percent sequence identity may be calculated as the number of identical residues as a percentage of the total residues in the SEQ ID NO referred to. The term “fragment” as used herein refers to a variant sequence that is a portion of a full-length polypeptide or polynucleotide. Fragments are typically selected regions of the polypeptide or polynucleotide that is of interest either functionally or, for example, in an assay.

[0451] Such variants, derivatives, homologues and fragments may be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5’ and 3’ flanking regions corresponding to the naturally-occurring sequence either side of the insertion site may be made. The flanking regions will contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed in accordance with the invention to make the encoded protein. These methods are only illustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used.

[0452] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example: Skoog, D.A., et al. (2013) Fundamentals of Analytical Chemistry, 9th edition, Cengage learning; Walker J.M. (2009) The Protein Protocols Handbook, 3rd edition, Springer Nature; Green, M.R. and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press; Ausubel, F.M., et al. (2003) Current Protocols in Molecular Biology, John Wiley & Sons; Hill, A. J. (2013) DNA Sequencing Protocols, Humana Press; Nielsen, B.S. and Jones, J. (2021 ) In Situ Hybridization Protocols, Springer US; Herdewijn, P. (2010) Oligonucleotide Synthesis: Methods and Applications, Humana Press; and Luo, Y. (2019) CRISPR Gene Editing: Methods and Protocols, Springer New York. Each of these general texts is herein incorporated by reference.

[0453] EXAMPLES The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

[0454] Example 1 - application of anion-exchange depth filter for an enhanced AAV manufacturing process

[0455] Purification of AAV vectors from cell culture is a critical step in the manufacturing process.

[0456] There are commercially-available depth filters which can be used for the clarification step. However, these depth filters come with different media and pore sizes and often involve pre-treatment, multiple time-consuming and expensive further purification steps, posing challenges in achieving clarification with high purity and yield of AAV vectors.

[0457] A typical AAV3 vector manufacturing process begins with the lysis of a HEK293 cell culture containing AAV3 vector particles. Subsequently, the lysate is passed through a depth filter using pump, tubes, and pressure sensors. The filtrate is collected and subjected to further purification, involving chromatography and tangential flow filtration.

[0458] Several commercially-available depth filters were trialled to assess their effectiveness:

[0459] • A fibrous anion exchange depth filter (3M Harvest RC depth filter) - comprising three primary components: four layers of anion exchange nonwoven having decreasing nominal pore size; a microporous membrane; and a membrane support. The anion exchange media is composed of polypropylene nonwovens that have been surface functionalized with a covalently attached quaternary ammonium polymer. The membrane is asymmetric polyethersulfone (PES) nominally rated at 0.2 pm. This is followed by a non-functionalized polypropylene nonwoven used as a support layer in the capsule.

[0460] • Depth filters comprised of cellulose fibers, filter aids (diatomaceous earth and / or perlite), and resins (PDP8 and V100P depth filters, available from PALL). The Seitz HP PDP8 dual layer filter is made up from a Seitz T1500P upstream, coarse layer and a Seitz K700P downstream, finer layer. • A depth filter comprising a synthetic filter media composed of silica filter aid, polyacrylic fiber and non-woven material (COSP depth filter, available from Merck).

[0461] • A depth filter comprising pleated, charged glass fiber layers (GF Plus depth filter, available from Sartopure)

[0462] Through the use of the fibrous anion exchange depth filter, the turbidity and hcDNA levels were reduced, leading to enhanced purity and yield of the AAV3 vector particles. The filtrate obtained from the fibrous anion exchange depth filter displayed lower precolumn pressure, indicating its compatibility and safety for use with the affinity chromatography column. Moreover, this filter demonstrated scalability, making it suitable for large-scale production.

[0463] Overall, the incorporation of a fibrous anion exchange depth filter in the AAV3 manufacturing process marks a significant advancement in gene therapy production.

[0464] Purification

[0465] The AAV-containing cell culture lysate is filtered through a fibrous anion exchange filter (here a 3M Harvest RC filter) at a flux of 100 LMH (LMH is L / hr / m2).

[0466] By effectively reducing turbidity and hcDNA levels, the fibrous anion exchange depth filter significantly improves the purity and yield of AAV3 vector particles (here AAV- LK03 vector particles which are AAV3-like vector particles). The clarified lysate serves as a cleaner and more refined starting material for subsequent purification steps.

[0467] Table 1 shows that the turbidity of the filtrates using the fibrous anion exchange depth filter and two other commercially available depth filters was similar immediately after filtration (see Filtrate Turbidity).

[0468] To further assess the purity of the filtrates, the turbidity was also measured following acidification. After the filtrate samples were acidified, the turbidity of the filtrate from the fibrous anion exchange depth filter remained low, while the turbidity of the filtrates from the other two filters increased significantly (see Filtrate Turbidity following sample acidification). This is because low pH can cause the aggregation of hcDNA and host cell proteins (HCPs) that remain in the solution, which results in higher turbidity. The data from Table 1 suggests that the fibrous anion exchange depth filter reduces the turbidity of the filtrate by reducing the levels of hcDNA and HCPs in the HEK293 cell culture lysate used for AAV3 vector production.

[0469] Table 1: Turbidity measurement of unclarified lysate, filtrate of fibrous anion exchange (AEX) depth filter, filtrate of PDP8+ V1 OOP+GF plus filter train, and filtrate of COSP+GF plus filter train.

[0470] Table 2 show the concentration of dsDNA in cell culture lysate as measured by PicoGreen assay. This shows that the fibrous anion exchange depth filter partly reduces amount of dsDNA from the HEK293 cell culture lysate for production of AAV3 vector particles.

[0471] Table 2: Quantification of dsDNA in cell culture lysate and fibrous anion exchange filtrate in AA V3 manufacturing process.

[0472] Product recovery Table 3 presents the quantitative analysis of AAV3 vector particles during the clarification step using ELISA for AAV capsid and ddPCR for vector genome. The data demonstrate nearly 100% recovery of the AAV3 vector particles, while preserving its genomic DNA, when employing a fibrous anion exchange depth filter in the clarification process. This shows that capsid recovery using the fibrous anion exchange depth filter is maintained.

[0473] Table 3: AA V3 vector recovery as measured by capsid ELISA and ddPCR.

[0474] Downstream processing

[0475] The reduced turbidity and hcDNA content in the lysate streamlined subsequent purification steps, simplifying the overall downstream processing. For example, when the fibrous anion exchange depth filter is used, no low pH precipitation is needed to eliminate impurities from the lysate prior to filtration. This results in a more efficient and cost-effective AAV manufacturing process.

[0476] The chromatograms in Figure 1 show the precolumn pressure in affinity chromatography as the load volume increases. The precolumn pressure is caused by the particulate matter in the load (Filtrate), which creates more resistance to flow as the load volume increases. The filtrate from the fibrous anion exchange depth filter (see Figure 1A), has a lower particulate matter content than the filtrate from other depth filters (see Figures 1 B-D), so it causes less resistance to flow and results in lower and steady precolumn pressure.

[0477] This suggests that the filtrate from the fibrous anion exchange depth filter is safer for the affinity column because it is less likely to cause column damage or clogging. The particulate matter in other depth filtrate (clarified lysate) could block the binding sites on the affinity column, leading to a decrease in binding capacity or process failure. It could also damage the column and pose safety risk.

[0478] Example 2 - application of two anion-exchange depth filters for an enhanced AAV manufacturing process

[0479] To further assess the impact of additional anion-exchange depth filters during purification, we performed the same purification method described in Example 1 , but using an additional anion-exchange depth filter to measure clarification of two separate batches of AAV3-like vector particles (here AAV-LK03 vector particles) with different genes of interest (GOI1 and GOI2).

[0480] We chose to use the 3M Harvest RC Centrate depth filter as the additional filter. The 3M Harvest RC Centrate is a fibrous anion exchange depth filter comprising multiple layers of quaternary amine functional anion exchange nonwoven media, a microporous asymmetric polyamide membrane functionalised with quaternary ammonium nominally rated at 0.2 pm for fine particulate removal (see product-info-sheet-pf-bf-en.pdf) and a non-functionalized polypropylene nonwoven used as a support layer. This depth filter is designed to be used with pre-clarified lysate, that is, post-centrifugation.

[0481] Purification of AAV3-like vector particles containing GOI1

[0482] The unclarified AAV-containing cell culture lysate is first filtered through a Harvest RC filter at a load of 40 L / m2and a flux of 33 LMH (LMH is L / hr / m2). The clarified filtrate is then passed through a Harvest RC Centrate filter, at a load of 100 L / m2and a flux of 100 LMH. The AAV is collected in the flow-through (effluent) of the Harvest RC Centrate step.

[0483] Table 4: Turbidity measurement of unclarified lysate, filtrate of fibrous anion exchange (AEX) depth filter (Harvest RC only), filtrate of AEX depth filter (Harvest RC and Harvest RC Centrate).

[0484] Product recovery

[0485] Table 5 presents the quantitative analysis of AAV3 vector particles containing gene of interest 1 (GOI1 ) during the clarification step using ddPCR to measure the vector genome. The data demonstrate approximately 87% vector genome recovery of the

[0486] AAV3 vector particles, when employing two fibrous anion exchange depth filters in sequence in the clarification process. This shows that vector recovery using the fibrous anion exchange depth filter is maintained.

[0487] Table 5: AA V3 vector recovery as measured by ddPCR per mL of clarified lysate.

[0488] Purification of AAV3-like vector particles containing GOI2

[0489] The unclarified AAV-containing cell culture lysate is first filtered through a Harvest RC filter at a load of 40 L / m2and a flux of 33 LMH. The clarified filtrate is then passed through a Harvest RC Centrate filter, at a load of 100 L / m2and a flux of 100 LMH. The AAV is collected in the flow-through (effluent) of the Harvest RC Centrate step.

[0490] Table 6 Turbidity measurement of unclarified lysate, filtrate of fibrous anion exchange (AEX) depth filter (Harvest RC only), filtrate of AEX depth filter (Harvest RC and Harvest RC Centrate).

[0491] Product recovery

[0492] Table 7 presents the quantitative analysis of AAV3 vector particles containing gene of interest 1 (GOI2) during the clarification step using qPCR to measure the vector genome. The data demonstrate approximately 90% vector genome recovery of the AAV3 vector particles, when employing two fibrous anion exchange depth filters in sequence in the clarification process. This shows that vector recovery using the fibrous anion exchange depth filter is maintained.

[0493] Table 7: AA V3 vector recovery as measured by qPCR per mL of clarified lysate.

[0494] By further reducing turbidity levels, use of an additional fibrous anion exchange depth filter significantly improves the purity and yield of AAV3 vector particles. The clarified lysate serves as a cleaner and more refined starting material for subsequent purification steps.

[0495] EMBODIMENTS

[0496] Various preferred features and embodiments of the present invention will now be described with reference to the following numbered paragraphs (paras).

[0497] 1 . A method for clarifying a cell culture, lysate or supernatant comprising AAV3 vector particles, the method comprising:

[0498] (a) providing a cell culture, lysate or supernatant comprising AAV3 vector particles; and (b) filtering the cell culture, lysate or supernatant through a depth filter to provide a clarified cell culture, lysate or supernatant, wherein the depth filter comprises a first filtration medium comprising an anion exchange nonwoven substrate comprising a plurality of quaternary ammonium groups.

[0499] 2. The method according to para 1 , wherein the depth filter comprises a fluid inlet, a fluid outlet, and one or more filtration mediums fluidly connecting the fluid inlet and the fluid outlet and contained in the same housing.

[0500] 3. The method according to para 1 or 2, wherein the depth filter comprises a first filtration medium comprising an anion exchange nonwoven substrate comprising a plurality of quaternary ammonium groups and a second filtration medium comprising a microporous membrane, wherein the first filtration medium is positioned upstream of the second filtration medium.

[0501] 4. The method according to any preceding para, wherein the depth filter comprises one or more layers, two or more layers, three or more layers, or four or more layers of the first filtration medium.

[0502] 5. The method according to any preceding para, wherein the first filtration medium comprises at least 0.1 mmol of quaternary ammonium groups per gram of the first filtration medium.

[0503] 6. The method according to any preceding para, wherein the quaternary ammonium groups have the formula -N+R1R2R3X wherein each of R1, R2, and R3are selected from methyl, ethyl, propyl and butyl groups, and wherein X’ is Cl’.

[0504] 7. The method according to any preceding para, wherein the quaternary ammonium groups are grafted via a linking group directly onto the nonwoven substrate.

[0505] 8. The method according to any preceding para, wherein the first filtration medium comprises polypropylene nonwovens that have been surface functionalized with a covalently attached quaternary ammonium polymer.

[0506] 9. The method according to any preceding para, wherein the first filtration medium has a thickness of from 0.1 mm to 10 mm. 10. The method according to any preceding para, wherein the anion exchange nonwoven substrate has an effective fibre diameter of from 1 micrometer to 6 micrometers.

[0507] 11 . The method according to any of paras 3-10, wherein the microporous membrane has a mean flow pore size of from 0.1 micrometer to 5 micrometers.

[0508] 12. The method according to any of paras 3-11 , wherein the microporous membrane has an asymmetric pore structure.

[0509] 13. The method according to any of paras 3-13, wherein the microporous membrane is from 5 micrometers to 800 micrometers thick.

[0510] 14. The method according to any of paras 3-13, wherein the microporous membrane is formed from polyethersulfone.

[0511] 15. The method according to any preceding para, wherein the depth filter further comprises a membrane support.

[0512] 16. The method according to para 15, wherein the membrane support is formed from polypropylene nonwoven.

[0513] 17. The method according to any preceding para, wherein the filtering of the cell culture, lysate or supernatant through the depth filter is performed at a flux rate of from 1 L / hr / m2to 300 L / hr / m2, preferably about 100 L / hr / m2

[0514] 18. The method according to any preceding para, wherein the filtering of the cell culture, lysate or supernatant through the depth filter is performed with a differential pressure of from 0.1 bar to 3.0 bar, preferably from 0.1 bar to 1 .5 bar.

[0515] 19. The method according to any preceding para, wherein the filtering of the cell culture, lysate or supernatant through the depth filter is performed with a feed volume of from 0.1 L to 5000L, preferably about 200L or about 500L.

[0516] 20. The method according to any preceding para, wherein the filtering of the cell culture, lysate or supernatant through the depth filter is performed at a temperature of from 2°C to 40°C, preferably from 15°C to 40°C. 21. A method for providing a composition comprising isolated AAV3 vector particles, the method comprising:

[0517] (a) providing a cell culture, lysate or supernatant comprising AAV3 vector particles;

[0518] (b) clarifying the cell culture, lysate or supernatant by the method according to any of paras 1 -20 to provide a clarified cell culture, lysate or supernatant; and

[0519] (c) isolating the AAV3 vector particles from the clarified cell culture, lysate or supernatant to provide a composition comprising isolated AAV3 vector particles.

[0520] 22. A method for providing a pharmaceutical composition comprising AAV3 vector particles, the method comprising:

[0521] (a) providing a cell culture, lysate or supernatant comprising AAV3 vector particles;

[0522] (b) clarifying the cell culture, lysate or supernatant by the method according to any of paras 1 -20 to provide a clarified cell culture, lysate or supernatant;

[0523] (c) isolating the AAV3 vector particles from the clarified cell culture, lysate or supernatant to provide a composition comprising isolated AAV3 vector particles; and

[0524] (d) formulating the composition comprising isolated AAV3 vector particles with one or more pharmaceutically acceptable carrier, diluent and / or excipient to provide a pharmaceutical composition comprising AAV3 vector particles.

[0525] 23. The method according to para 22, wherein the method further comprises a step

[0526] (e) of aseptic filling the pharmaceutical composition into a sterile container and sealing the container.

[0527] 24. The method according to any of paras 21 to 23, wherein the AAV3 vector particles are isolated by one or more of tangential flow filtration, normal flow filtration, affinity chromatography, size exclusion chromatography, ion exchange chromatography, mixed mode chromatography and hydrophobic interaction chromatography. 25. The method according to any of paras 21 to 24, wherein the AAV3 vector particles are isolated by affinity chromatography.

[0528] 26. The method according to any of paras 21 to 25, wherein the method further comprises a step of separating empty AAV3 capsids from full AAV3 capsids.

[0529] 27. The method according to any of paras 21 to 26, wherein the method further comprises one or more steps of sterile filtration.

[0530] 28. The method according to any preceding para, wherein the AAV3 vector particles comprise an AAV capsid protein having at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: #1# or SEQ ID NO: #2#.

[0531] 29. The method according to any preceding para, wherein the AAV3 vector particles comprise an AAV-LK03 capsid protein.

[0532] 30. The method according to any preceding para, wherein the AAV3 vector particles comprise an AAV capsid protein having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: #3#.

[0533] 31 . The method according to any preceding para, wherein the AAV3 vector particles comprise an AAV capsid protein having at least 99.0%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity to the amino acid sequence of SEQ ID NO: #3#.

[0534] 32. The method according to any preceding para, wherein the AAV3 vector particles comprise an AAV capsid protein having the amino acid sequence of SEQ ID NO: #3#.

[0535] 33. The method according to any preceding para, wherein the AAV3 vector particles comprise an AAV genome that is a derivative of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 , optionally wherein the AAV genome is a derivative of AAV2.

[0536] 34. The method according to any preceding para, wherein the AAV3 vector particles encode a therapeutic gene product. 35. The method according to any preceding para, wherein the AAV3 vector particles encode a polypeptide associated with a kidney disease, optionally wherein the AAV3 vector particles encode a NPHS2, CFI, CFH, FHL-1 , COL4A3, COL4A4, COL4A5, C1 INH, C4BP, MASP2, C3, C5aR1 , C5, C5a, CD55, CD35, CD46, CD59, vitronectin, clusterin, ADCK4, ALG1 , ARHGAP24, ARGHDIA, CD151 , CD2AP, C0Q2, C0Q6, DGKE, E2F3, EMP2, KANK2, LAGE3, LMNA, LMX1 B, MAF B, NUP85, NUP93, NXF5, OSGEP, PAX2, PDSS2, PMM2, PODXL, SCARB2, SGPL1 , Smad7, TP53RK, TPRKB, VDR, WDR73, WT1 , ZMPSTE24, APOL1 , NPHS1 , TRPC6, NUP107, NUP133, NUP160, ACTN4, INF2, ANKFY1 , ANLN, CRB2, ITGA3, KANK1 , KANK4, MAGI2, MY01 E, OCRL, PTPRO, SMARCAL1 , SYNPO, TBC1 D8B, XP05, TNS2, NLRP3, or VEGFC polypeptide.

[0537] 36. The method according to any preceding para, wherein the cell culture, lysate or supernatant is a cell culture.

[0538] 37. The method according to any preceding para, wherein the cell culture comprises AAV producer and / or packaging cells, preferably wherein the AAV producer and / or packaging cells are HEK293, HEK293T, COS-1 , COS-7, CV-1 , HeLa, CHO, SF-9 or A549 cells, or derivatives thereof, more preferably wherein the AAV producer and / or packaging cells are HEK293 cells or derivatives thereof.

[0539] 38. The method according to any preceding para, wherein the cell culture has a cell density of from 1x106cells / mL to 5x107cells / mL.

[0540] 39. The method according to any preceding para, wherein the cell culture, lysate or supernatant is a cell lysate.

[0541] 40. The method according to any preceding para, wherein the cell lysate comprises lysed AAV producer and / or packaging cells, preferably wherein the AAV producer and / or packaging cells are HEK293, HEK293T, COS-1 , COS-7, CV-1 , HeLa, CHO, SF-9 or A549 cells, or derivatives thereof, more preferably wherein the AAV producer and / or packaging cells are HEK293 cells or derivatives thereof.

[0542] 41 . The method according to any preceding para, wherein the cell lysate is derived from a cell culture that has a cell density before lysing of from 1x106cells / mL to 5x107cells / mL. 42. The method according to any preceding para, wherein the cell culture, lysate or supernatant is a cell supernatant.

[0543] 43. The method according to any preceding para, wherein the cell culture, lysate or supernatant is not treated with endonuclease.

[0544] 44. The method according to any preceding para, wherein the cell culture, lysate or supernatant comprises the AAV3 vector particles in an amount of from 1x1011capsids / mL to 5x1013capsids / mL, optionally determined by ELISA.

[0545] 45. The method according to any preceding para, wherein the cell culture, lysate or supernatant comprises the AAV3 vector particles in an amount of from 1x1010vg / mL to 5x1012vg / mL, optionally determined by ddPCR.

[0546] 43. The method according to any preceding para, wherein the clarified cell culture, lysate or supernatant has a turbidity of 50 NTU or less in an acidification assay, optionally wherein the turbidity is determined by a turbidimeter.

[0547] 44. The method according to any preceding para, wherein the clarified cell culture, lysate or supernatant has a dsDNA concentration of 0.5 mg / L or less, 0.4 mg / L or less, 0.3 mg / L or less, 0.2 mg / L or less, or 0.1 mg / L or less, optionally wherein the dsDNA concentration is determined based on the fluorescence intensity of fluorescent dye binding to dsDNA using a fluorometer.

[0548] 45. The method according to any preceding para, wherein the clarified cell culture, lysate or supernatant comprises the AAV3 vector particles in an amount of from 1x1011capsids / mL to 5x1013capsids / mL, optionally determined by ELISA.

[0549] 46. The method according to any preceding para, wherein the clarified cell culture, lysate or supernatant has a capsid recovery rate of the AAV3 vector particles of 80% or more, optionally determined by ELISA.

[0550] 47. The method according to any preceding para, wherein the clarified cell culture, lysate or supernatant comprises the AAV3 vector particles in an amount of from 1x101° vg / mL to 5x1012vg / mL, optionally determined by ddPCR. 48. The method according to any preceding para, wherein the clarified cell culture, lysate or supernatant has a vector genome recovery rate of the AAV3 vector particles of 80% or more, optionally determined by ddPCR.

[0551] 49. The method according to any preceding para, wherein the method does not comprise any other clarification steps.

[0552] 50. The method according to any preceding para, wherein the method does not comprise any other filtration steps during clarification.

[0553] 51 . A clarified cell culture, lysate or supernatant obtained or obtainable by the method of any of paras 1 -20 or 28-50.

[0554] 52. A composition comprising isolated AAV vector particles obtained or obtainable by the method of any of paras 21 or 24-50.

[0555] 53. A pharmaceutical composition comprising isolated AAV vector particles obtained or obtainable by the method of any of paras 22-50.

[0556] 54. Use of a depth filter for clarifying a cell culture, lysate or supernatant comprising AAV3 vector particles, wherein the depth filter comprises a first filtration medium comprising an anion exchange nonwoven substrate comprising a plurality of quaternary ammonium groups.

[0557] 55. The use according to para 54, wherein the depth filter is defined according to any of paras 2-16.

[0558] 56. The use according to para 54 or 55, wherein the AAV3 vector particles are defined according to any of paras 28-35.

[0559] 57. The use according to any of paras 54-56, wherein the cell culture, lysate or supernatant is defined according to any of paras 36-42.

[0560] 58. The use according to any of paras 54-57, wherein the clarified cell culture, lysate or supernatant is defined according to any of paras 43-48.

Claims

CLAIMS1 . A method for clarifying a cell culture, lysate or supernatant comprising AAV3 vector particles, the method comprising:(a) providing a cell culture, lysate or supernatant comprising AAV3 vector particles; and(b) filtering the cell culture, lysate or supernatant through a first depth filter to provide a clarified cell culture, lysate or supernatant, wherein the first depth filter comprises a first filtration medium comprising an anion exchange nonwoven substrate comprising a plurality of quaternary ammonium groups.

2. The method according to claim 1 , wherein a second depth filter is used in combination with the first depth filter.

3. The method according to claim 2, comprising the further step of:(c) filtering the clarified cell culture, lysate or supernatant from step (b) through a second depth filter to provide a further clarified cell culture, lysate or supernatant, optionally wherein the second depth filter comprises a first filtration medium comprising an anion exchange nonwoven substrate comprising a plurality of quaternary ammonium groups.

4. The method according to any preceding claim, wherein the depth filter(s) comprise(s) a fluid inlet, a fluid outlet, and one or more filtration mediums fluidly connecting the fluid inlet and the fluid outlet and contained in the same housing, preferably wherein the depth filter comprises a first filtration medium comprising an anion exchange nonwoven substrate comprising a plurality of quaternary ammonium groups and a second filtration medium comprising a microporous membrane, wherein the first filtration medium is positioned upstream of the second filtration medium, optionally wherein the depth filter comprises one or more layers, two or more layers, three or more layers, or four or more layers of the first filtration medium.

5. The method according to any preceding claim, wherein the first filtration medium comprises at least 0.1 mmol of quaternary ammonium groups per gram of the first filtration medium,preferably wherein the quaternary ammonium groups have the formula -N+R1R2R3X’ , wherein each of R1, R2, and R3are selected from methyl, ethyl, propyl and butyl groups, and wherein X’ is Cl; optionally wherein the quaternary ammonium groups are grafted via a linking group directly onto the nonwoven substrate, preferably wherein the first filtration medium comprises polypropylene nonwovens that have been surface functionalized with a covalently attached quaternary ammonium polymer.

6. The method according to any preceding claim, wherein the first filtration medium has a thickness of from 0.1 mm to 10 mm, and / or wherein the anion exchange nonwoven substrate has an effective fibre diameter of from 1 micrometer to 6 micrometers.

7. The method according to any of claims 4-6, wherein the microporous membrane: (a) has a mean flow pore size of from 0.1 micrometer to 5 micrometers; (b) has an asymmetric pore structure; (c) is from 5 micrometers to 800 micrometers thick; and / or (d) is formed from polyethersulfone.

8. The method according to any preceding claim, wherein the depth filter(s) further comprises a membrane support, optionally wherein the membrane support is formed from polypropylene nonwoven.

9. The method according to any preceding claim, wherein the filtering of the cell culture, lysate or supernatant through the depth filter(s) is performed: (a) at a flux rate of from 1 L / hr / m2to 300 L / hr / m2, preferably from 33 L / hr / m2to 100 L / hr / m2preferably about 100 L / hr / m2or preferably about 33 L / hr / m2; (b) with a differential pressure of from 0.1 bar to 3.0 bar, preferably from 0.1 bar to 1.5 bar; (c) with a feed volume of from 0.1 L to 5000L, preferably about 200L or about 500L; and / or (d) at a temperature of from 2°C to 40°C, preferably from 15°C to 40°C.

10. The method according to any preceding claim, wherein the method does not comprise a step of binding the AAV3 vector particles to the depth filter(s) and / or does not comprise a step of eluting the AAV3 vector particles from the depth filter(s).

11. A method for providing a composition comprising isolated AAV3 vector particles, the method comprising:(a) providing a cell culture, lysate or supernatant comprising AAV3 vector particles;(b) clarifying the cell culture, lysate or supernatant by the method according to any of claims 1 -10 to provide a clarified cell culture, lysate or supernatant; and(c) isolating the AAV3 vector particles from the clarified cell culture, lysate or supernatant to provide a composition comprising isolated AAV3 vector particles.

12. A method for providing a pharmaceutical composition comprising AAV3 vector particles, the method comprising:(a) providing a cell culture, lysate or supernatant comprising AAV3 vector particles;(b) clarifying the cell culture, lysate or supernatant by the method according to any of claims 1 -10 to provide a clarified cell culture, lysate or supernatant;(c) isolating the AAV3 vector particles from the clarified cell culture, lysate or supernatant to provide a composition comprising isolated AAV3 vector particles;(d) formulating the composition comprising isolated AAV3 vector particles with one or more pharmaceutically acceptable carrier, diluent and / or excipient to provide a pharmaceutical composition comprising AAV3 vector particles; and(e) optionally, aseptic filling the pharmaceutical composition into a sterile container and sealing the container.

13. The method according to claim 11 or 12, wherein the AAV3 vector particles are isolated by one or more of tangential flow filtration, normal flow filtration, affinity chromatography, size exclusion chromatography, ion exchange chromatography, mixed mode chromatography and hydrophobic interaction chromatography, preferably wherein the AAV3 vector particles are isolated by affinity chromatography, optionally wherein the method further comprises a step of separating empty AAV3 capsids from full AAV3 capsids and / or the method further comprises one or more steps of sterile filtration.

14. The method according to any preceding claim, wherein the AAV3 vector particles are: AAV3B vector particles, AAV3A vector particles, AAV-LK03 vector particles, AAV3B-DE5 vector particles, AAV.GT5 vector particles, AAV3-ST vector particles, AAV3B-V04 vector particles, AAV3B-V05 vector particles, AAV3B-G3 vector particles, AAV3B-E12 vector particles, AAV-KP1 vector particles, AAV-KP2 vector particles, AAV-KP3 vector particles or variants thereof.

15. The method according to any preceding claim, wherein the AAV3 vector particles comprise an AAV capsid protein having at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: #1# or SEQ ID NO: #2#.

16. The method according to any preceding claim, wherein the AAV3 vector particles comprise an AAV-LK03 capsid protein, preferably wherein the AAV3 vector particles comprise an AAV capsid protein having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: #3#, more preferably wherein the AAV3 vector particles comprise an AAV capsid protein having at least 99.0%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity to the amino acid sequence of SEQ ID NO: #3#, most preferably wherein the AAV3 vector particles comprise an AAV capsid protein having the amino acid sequence of SEQ ID NO: #3#.

17. The method according to any preceding claim, wherein the AAV3 vector particles comprise an AAV genome that is a derivative of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 , optionally wherein the AAV genome is a derivative of AAV2.

18. The method according to any preceding claim, wherein the AAV3 vector particles encode a therapeutic gene product, preferably wherein the AAV3 vector particles encode a polypeptide associated with a kidney disease, optionally wherein the AAV3 vector particles encode a NPHS2, CFI, CFH, FHL-1 , COL4A3, COL4A4, COL4A5, C1 INH, C4BP, MASP2, C3, C5aR1 , C5,C5a, CD55, CD35, CD46, CD59, vitronectin, clusterin, ADCK4, ALG1 , ARHGAP24, ARGHDIA, CD151 , CD2AP, C0Q2, C0Q6, DGKE, E2F3, EMP2, KANK2, LAGE3, LMNA, LMX1 B, MAF B, NUP85, NUP93, NXF5, OSGEP, PAX2, PDSS2, PMM2, PODXL, SCARB2, SGPL1 , Smad7, TP53RK, TPRKB, VDR, WDR73, WT1 , ZMPSTE24, APOL1 , NPHS1 , TRPC6, NUP107, NUP133, NUP160, ACTN4, INF2, ANKFY1 , ANLN, CRB2, ITGA3, KANK1 , KANK4, MAGI2, MY01 E, OCRL, PTPRO, SMARCAL1 , SYNPO, TBC1 D8B, XPO5, TNS2, NLRP3, or VEGFC polypeptide.

19. The method according to any of claims 1 -18, wherein the cell culture, lysate or supernatant is a cell culture, optionally wherein the cell culture comprises AAV producer and / or packaging cells, preferably wherein the AAV producer and / or packaging cells are HEK293, HEK293T, COS-1 , COS-7, CV-1 , HeLa, CHO, SF-9 or A549 cells, or derivatives thereof, more preferably wherein the AAV producer and / or packaging cells are HEK293 cells or derivatives thereof, and / or the cell culture has a cell density of from 1x106cells / mL to 5x107cells / mL.

20. The method according to any of claims 1 -18, wherein the cell culture, lysate or supernatant is a cell lysate, optionally wherein the cell lysate comprises lysed AAV producer and / or packaging cells, preferably wherein the AAV producer and / or packaging cells are HEK293, HEK293T, COS-1 , COS-7, CV-1 , HeLa, CHO, SF-9 or A549 cells, or derivatives thereof, more preferably wherein the AAV producer and / or packaging cells are HEK293 cells or derivatives thereof, and / or the cell lysate is derived from a cell culture that has a cell density before lysing of from 1x106cells / mL to 5x107cells / mL.

21. The method according to any of claims 1 -18, wherein the cell culture, lysate or supernatant is a cell supernatant.

22. The method according to any preceding claim, wherein the cell culture, lysate or supernatant is not treated with endonuclease.

23. The method according to any preceding claim, wherein the cell culture, lysate or supernatant: (a) comprises the AAV3 vector particles in an amount of from 1x1011capsids / mL to 5x1013capsids / mL, optionally determined by ELISA; and / or (b) comprises the AAV3 vector particles in an amount of from 1x1010vg / mL to 5x1012vg / mL, optionally determined by ddPCR.

24. The method according to any preceding claim, wherein the clarified cell culture, lysate or supernatant: (a) has a turbidity of 50 NTU or less, optionally wherein the turbidity is determined by a turbidimeter; (b) has a dsDNA concentration of 0.5 mg / L or less, 0.4 mg / L or less, 0.3 mg / L or less, 0.2 mg / L or less, or 0.1 mg / L or less, optionally wherein the dsDNA concentration is determined based on the fluorescence intensity of fluorescent dye binding to dsDNA using a fluorometer; (c) comprises the AAV3 vector particles in an amount of from 1x1011capsids / mL to 5x1013capsids / mL, optionally determined by ELISA; (d) has a capsid recovery rate of the AAV3 vector particles of 80% or more, optionally determined by ELISA; (e) comprises the AAV3 vector particles in an amount of from 1x101° vg / mL to 5x1012vg / mL, optionally determined by ddPCR; (f) has a vector genome recovery rate of the AAV3 vector particles of 80% or more, optionally determined by ddPCR; and / or (g) has a lower turbidity than the unclarified cell culture, lysate or supernatant..

25. The method according to any preceding claim, wherein the method does not comprise any other clarification steps.

26. A clarified cell culture, lysate or supernatant obtained or obtainable by the method of any of claims 1 -10 or 14-25.

27. A composition comprising isolated AAV vector particles obtained or obtainable by the method of any of claims 11 or 13-25.

28. A pharmaceutical composition comprising isolated AAV vector particles obtained or obtainable by the method of any of claims 12-25.

29. Use of a depth filter for clarifying a cell culture, lysate or supernatant comprising AAV3 vector particles, wherein the depth filter comprises a first filtration medium comprising an anion exchange nonwoven substrate comprising a plurality of quaternary ammonium groups,optionally wherein: (a) the depth filter is defined according to any of claims 1-8; the AAV3 vector particles are defined according to any of claims 414-18; the cell culture, lysate or supernatant is defined according to any of claims 19-23 and / or the clarified cell culture, lysate or supernatant is defined according to claim 24.

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