WO2025217029A1 - Compositions & methods for adeno-associated virus capsid variant purification - Google Patents

Abstract

Described and provided herein are compositions and methods for the differential separation of recombinant adeno-associated viral capsid variants, wherein the methods comprise at least two chromatography polishing steps, e.g., cation exchange chromatography steps, anion exchange chromatography steps, hydrophobic interaction chromatography steps, and/or mixed mode chromatography steps.

Description

COMPOSITIONS & METHODS FOR ADENO-ASSOCIATED VIRUS CAPSID VARIANT PURIFICATION

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/575,763, filed April 7, 2024, the contents of which are incorporated by reference in their entirety, and to which priority is claimed.

INTRODUCTION

Gene delivery is a promising method for the treatment of acquired and inherited diseases. A number of viral-based systems for gene transfer purposes have been described, including adeno- associated virus (AAV)-based systems and recombinant adeno-associated virus (rAAV)-based systems.

AAV is a helper-dependent DNA parvovirus that belongs to the genus Dependovirus. AAV requires helper virus function, e.g., adenovirus, herpes virus, or vaccinia, in order for a productive infection to occur. In the absence of a helper virus functions, AAV establishes a latent state by inserting its genome into a host cell chromosome. Subsequent infection by a helper virus rescues the integrated viral genome, which can then replicate to produce infectious AAV progeny.

AAV and rAAV-based systems have a wide host range and are able to replicate in cells from any species in the presence of a suitable helper virus. For example, human AAV will replicate in canine cells co-infected with a canine adenovirus. AAV has not been associated with any human or animal disease and does not appear to adversely affect the biological properties of the host cell upon integration. rAAV vectors can be engineered to carry a heterologous nucleic acid sequence of interest (e.g., a selected gene encoding a therapeutic protein, an inhibitory nucleic acid such as an antisense molecule, a ribozyme, a miRNA, etc.) by deleting, in whole or in part, the internal portion of the AAV genome and inserting the nucleic acid sequence of interest between the ITRs. The ITRs remain functional in such vectors allowing replication and packaging of the rAAV containing the heterologous nucleic acid sequence of interest. The heterologous nucleic acid sequence is also typically linked to a promoter sequence capable of driving expression of the nucleic acid in the patient's target cells. Termination signals, such as polyadenylation sites, can also be included in the vector.

The construction of infectious rAAV vectors has been described in a number of publications. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Numbers WO 92/01070 and WO 93/03769; Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Current Topics in Microbiol, and Immunol. 158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801. rAAV vectors have shown excellent therapeutic promise in several early phase clinical trials by multiple groups. Development of this new class of biologic product towards approval will involve improvements in vector characterization and quality control methods, including a better understanding of how vector design and manufacturing process parameters affect impurity profiles, including the presence of rAAV capsid variants, in clinical grade rAAV-based systems.

SUMMARY

The instant disclosure provides compositions and methods for the purification of rAAV vector capsids. For example, but not by way of limitation, certain rAAV purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants. In certain embodiments, the methods of the present disclosure comprise: (a) contacting a sample comprising rAAV vector particles to an AAV affinity chromatography composition to produce a capture step eluate comprising rAAV vector particles; (b) contacting said capture step eluate to a first polishing chromatography composition to produce a first polishing eluate comprising rAAV vector particles; and (c) contacting said first polishing eluate to a second polishing chromatography composition to produce a second polishing eluate comprising differentially separated rAAV vector capsid variants. In certain of such embodiments, the capture step eluate and/or the first polishing eluate is incubated in a buffer solution comprising about 50 mM to about 100 mM Tris at a pH of about 4.5 to about 9.0, and a conductivity of about 2.5 mS/cm to about 15 mS/cm. In certain embodiments, the incubation time is between about 1 hour to about 12 hours.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition and the second polishing chromatography composition are different.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is selected from the group consisting of: a strong anion exchange (AEX) chromatography composition; a mixed mode chromatography (MMC) composition; a strong cation exchange (CEX) chromatography composition; hydrophobic chromatography (HIC) composition; and a weak AEX chromatography composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the second polishing chromatography composition is selected from the group consisting of a weak AEX chromatography composition; a strong AEX chromatography composition; a HIC composition; and a MMC composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is selected from the group consisting of a strong AEX chromatography composition; a strong CEX chromatography composition; a HIC composition; a MMC composition; and a weak AEX chromatography composition; while the second polishing chromatography composition is selected from the group consisting of a weak AEX chromatography composition; a strong AEX chromatography composition; a hydrophobic interaction chromatography composition; and a MMC composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a strong AEX chromatography composition, and the second polishing chromatography composition is a weak AEX chromatography composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a first strong AEX chromatography composition, and the second polishing chromatography composition is a second, distinct, strong AEX chromatography composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a strong AEX chromatography composition, and the second polishing chromatography composition is a HIC composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a strong AEX chromatography composition, and the second polishing chromatography composition is a MMC composition. In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a strong CEX chromatography composition, and the second polishing chromatography composition is a weak AEX chromatography composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a strong CEX chromatography composition, and the second polishing chromatography composition is a strong AEX chromatography composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a strong CEX chromatography composition, and the second polishing chromatography composition is a HIC chromatography composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a strong CEX chromatography composition, and the second polishing chromatography composition is a MMC composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a MMC composition, and the second polishing chromatography composition is a weak AEX chromatography composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a MMC composition, and the second polishing chromatography composition is a strong AEX chromatography composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a MMC composition, and the second polishing chromatography composition is a HIC chromatography composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a HIC chromatography composition, and the second polishing chromatography composition is a weak AEX chromatography composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a HIC chromatography composition, and the second polishing chromatography composition is a strong AEX chromatography composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a HIC chromatography composition, and the second polishing chromatography composition is a MMC composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and a capsid variant is differentially separated to at least 50% purity. In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and a capsid variant is differentially separated to at least 60% purity. In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and a capsid variant is differentially separated to at least 70% purity. In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and a capsid variant is differentially separated to at least 80% purity. In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and a capsid variant is differentially separated to at least 90% purity. In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and a capsid variant is differentially separated to at least 95% purity.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the capture step eluate and/or the first polishing eluate is diluted with a Tris buffer, wherein said Tris buffer comprises:(i) about 50 mM to about 100 mM Tris; (ii) a pH of about 4.5 to about 9.0; and (iii) a conductivity of about 6 mS/cm to 16 mS/cm. In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and contacting of the first and/or second polishing chromatography composition comprises equilibrating and/or washing said composition with a Tris buffer, wherein said Tris buffer comprises: (i) about 50 mM to about 100 mM Tris; (ii) a pH of about 7.5 to about 9.0; and (iii) a conductivity of about 6 mS/cm to 16 mS/cm.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and said rAAV vector capsid variants are eluted from said first and/or second polishing chromatography composition by contacting said composition with a salt-based elution buffer comprising: (i) about 50 mM to about 100 mM Tris; (ii) a pH of about 7.5 to about 9.0; and (iii) a conductivity of about 10 mS/cm to about 30 mS/cm.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and said rAAV vector capsid variants are eluted from said first and/or second polishing chromatography composition by contacting said composition with a pH-based elution buffer comprising: (i) about 50 mM to 200 mM sodium acetate; (ii) a pH of about 3.0 to about 6.0; and (iii) a NaCl concentration of about 250 mM or greater.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants. In certain embodiments, an isolated capsid variant has a different relative potency in comparison to rAAV vector particles produced from a AAV affinity based capture step. In certain embodiments, an isolated capsid variant has a different relative potency in comparison to rAAV vector particles produced from a first polishing chromatography step. In certain embodiments, an isolated capsid variant has higher relative potency in comparison to rAAV vector particles produced from a AAV affinity based capture step. In certain embodiments, an isolated capsid variant has a higher relative potency in comparison to rAAV vector particles produced from a first polishing chromatography step.

In particular aspects of the instant methods, a method is performed according to any one or more column, condition, concentration, molarity, volume, capacity, material, pH, or step as set forth in any of the Examples included herein. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 depicts a representative high-performance liquid chromatography (HPLC) separation analysis of LK03 capsid variants disclosed herein.

Figures 2A-2C depict exemplary sequential chromatography polishing steps of strong anion exchange (AEX) —> weak AEX chromatography for the differential separation of rAAV capsid variants disclosed herein. Figure 2A depicts a separation of empty and several non-empty rAAV capsid variants using a Poros XQ chromatography composition. In particular, Figure 2A depicts the PXQ trace, from which Peaks 2(P2), 3(P3), 4(P4) and 5(P5) were collected. Peak 1 is empty capsids. Figure 2B depicts the differential separation of empty and several non-empty rAAV capsid variants using a Monolith BIA DEAE chromatographic composition onto which samples eluted from Poros XQ chromatographic composition, specifically the P2, P3, and P4 peaks, were contacted. The LK03 capsid relative potency and empty/partial/full% compared to the least potent peak (P2f) is listed in Figure 2C.

Figure 3 depicts exemplary sequential chromatography polishing steps of strong AEX (Poros XQ) —> strong AEX (BIA monolith QA) chromatography for the differential separation of rAAV capsid variants disclosed herein. Figure 3 depicts a separation of empty and several nonempty rAAV capsid variants using a Monolith BIA QA chromatography composition onto which samples eluted from the Poros XQ chromatography composition (as Peak 2) were contacted.

Figures 4A-4B depict exemplary sequential chromatography polishing steps of strong cation exchange (CEX) —> strong AEX chromatography for the differential separation of rAAV capsid variants disclosed herein. Figure 4A depicts a separation of empty and several non-empty rAAV capsid variants using a Poros XS chromatography composition. Figure 4B depicts a separation of empty and several non-empty rAAV capsid variants using a Poros XQ chromatography composition onto which samples eluted from the Poros XS chromatography composition (as Peak 2) were contacted.

Figure 5 depicts exemplary sequential chromatography polishing steps of strong AEX (Poros XQ) —> strong AEX (Monolith QA) chromatography for the differential separation of rAAV capsid variants disclosed herein. Figure 5 depicts a separation of empty and several non-empty rAAV capsid variants using a Monolith QA chromatography composition onto which samples eluted from the Poros XQ chromatography composition (as Peak 2) were contacted.

Figures 6A-6B depict exemplary sequential chromatography polishing steps of strong AEX (Poros XQ) —> strong AEX (membrane Q) chromatography for the differential separation of rAAV capsid variants disclosed herein. Figure 6A depicts a separation of empty and several non- empty rAAV capsid variants using a Sartobind Q chromatography composition onto which samples eluted from the Poros XQ chromatography composition (as Peak 2) were contacted. Figure 6B depicts a separation of empty and several non-empty rAAV capsid variants using a Mustang Q chromatography composition onto which samples eluted from the Poros XQ chromatography composition (as Peak 2) were contacted.

Figures 7A-7B depict exemplary sequential chromatography polishing steps of mixed mode (Capto Adhere anion and hydrophobic) resin — weak AEX (BIA monolith DEAE) chromatography for the differential separation of rAAV capsid variants disclosed herein. Figure 7A depicts a separation of empty and several non-empty capsid variants using a mixed mode Capto Adhere chromatography composition. Figure 7B depicts a separation of empty and several nonempty capsid variants on a BIA monolith DEAE chromatography composition onto which samples eluted from the Capto Adhere chromatography composition (as Peak 2) were contacted.

Figures 8A-8B depict exemplary sequential chromatography polishing steps of strong AEX (Poros XQ) —> hydrophobic interaction chromatography (HIC) for the differential separation of rAAV capsid variants disclosed herein. Figure 8A depicts a separation of empty and several non-empty rAAV capsid variants using a Capto Butyl ImpRes HIC chromatography composition onto which samples eluted from the Poros XQ chromatography composition (as Peak 2) were contacted. Figure 8B depicts a separation of empty and several non-empty rAAV capsid variants using a Capto Phenyl ImpRes HIC chromatography composition onto which samples eluted from the Poros XQ chromatography composition (as Peak 2) were contacted.

Figure 9 depicts exemplary sequential chromatography polishing steps of strong AEX (Poros XQ) —> mixed mode chromatography for the differential separation of rAAV capsid variants disclosed herein. Figure 9 depicts a separation of empty and several non-empty rAAV capsid variants using a Monolith Prima S chromatography composition onto which samples eluted from the Poros XQ chromatography composition (as Peak 2) were contacted.

Figures 10A-10C depict analytical scale comparisons of capsid heterogeneity recovered by various exemplary chromatography operations disclosed herein. The capsid of the AAV in Figures 1-10 is LK03.

Figures 11A-11B depict exemplary separation of RHM4-1 capsid variants using strong cation exchange (CEX) polishing step, when eluted with pH gradient (Figure 11 A) or salt gradient (Figure 1 IB). DETAILED DESCRIPTION

The present disclosure is directed to compositions and methods useful in reducing rAAV- containing product heterogeneity, enhancing rAAV-containing product consistency, and increasing the potency and stability of rAAVs. For example, but not by way of limitation, the presently disclosed subject matter provides rAAV purification processes. In certain embodiments, the present disclosure is directed to rAAV purification methods comprising a capture chromatography step, e.g., an affinity chromatography step, followed by a two-step chromatography polishing operation. In certain embodiments, the first of the two chromatography polishing steps is selected from the group consisting of: a strong AEX chromatography composition; a EUC composition; a MMC composition; a strong CEX chromatography composition; and a weak AEX chromatography composition. In certain embodiments, the second polishing chromatography composition is selected from the group consisting of: a weak AEX chromatography composition; a strong AEX chromatography composition; a HIC composition; a strong CEX chromatography composition; and a MMC composition. In certain embodiments, the methods disclosed herein comprising two sequential polishing steps result in increased product homogeneity and reduced product-related impurities such as the presence of differing capsid variants.

1. Definitions

The indefinite articles “a” and “an” refer to at least one of the associated noun, and are used interchangeably with the terms “at least one” and “one or more.” For example, “a module” means at least one module, or one or more modules.

Impurities include AAV vector production related impurities which include proteins, nucleic acids (e.g., DNA), cellular components such as intracellular and membrane components which are impurities distinct from the AAV vectors. The term "production or process related impurities" refers to any components released during the AAV purification and production process that are not bona fide rAAV particles.

Bona fide rAAV vectors refer to rAAV vector particles comprising the heterologous nucleic acid (e.g., transgene) which are capable of infecting target cells. The phrase excludes empty AAV capsids, AAV vectors lacking full inserts in the packaged genome or AAV vectors containing contaminating host cell nucleic acids. In certain embodiments, bona fide rAAV vectors refer to rAAV vector particles that also lack contaminating plasmid sequences in the packaged vector genome. "Empty capsids" and "empty particles" refer to an AAV particle or virion that includes an AAV capsid shell but that lacks the genome comprising the heterologous nucleic acid sequence flanked on one or both sides by AAV ITRs. Such empty capsids do not function to transfer the heterologous nucleic acid sequence into the host cell or cells within an organism.

“Partial capsids” and “partial particles” refer to an AAV particle or virion that includes an AAV capsid shell and contains part or a fragment of the genome comprising the heterologous nucleic acid sequence flanked on one or both sides by AAV ITRs. Such partial capsids do not effectively function to transfer the heterologous nucleic acid sequence into the host cell or cells within an organism.

“Full capsids” and “full particles” refer to an AAV particle or virion that includes an AAV capsid shell and contains the whole genome comprising the heterologous nucleic acid sequence flanked on one or both sides by AAV ITRs. Such full capsids effectively function to transfer the heterologous nucleic acid sequence into the host cell or cells within an organism.

Populations of AAV capsids are separated as “empty,” “partial,” or “full” by chromatography (e.g., ion-exchange chromatography, hydrophobic interaction chromatography) depending on their retention times.

As used herein the term, “capsid variants” refers not only to empty, partial, and full capsids, but also to other capsid variants that differ not in terms of the size of the encapsulated transgene but rather in the composition of the capsid itself. For example, but not by way of limitation, such capsids variants can comprise one or more modifications to one or more of capsid proteins VP1, VP2, and VP3. Such modifications to the capsid proteins can render the capsid: (i) less potent and/or (ii) unstable.

As used herein the term “potency” refers to the ability of a rAAV vector to deliver to a target cell, a heterologous nucleic acid sequence which is expressed as a transgene. The potency of a test sample (rAAV vector) can be assessed by cell-based assays by administering a fixed titer (Vg/mL) of the rAAV vector to the target cell. “Relative potency” refers to the ratio of the potency (presented in the form of percentage) for the test samples (for a given fixed titer (Vg/mL)) to that of a reference sample at the same titer (Vg/mL)). The reference sample can be the same rAAV molecule (e.g., capsid). The relative potency accounts for die inherent variability of cell-based assays, where absolute potency is less reliable.

“Chromatography” refers to a method for separating or purifying various components (e.g., proteins) from a mixture.

As used herein, “chromatography matrix” refers to a distinct chromatographic material with which proteins interact in the stationary phase through various modes or mechanism. Resins, ligands, membranes, and a pre-packaged manufactured column are non-limiting examples of chromatography matrices.

As used herein, “IEX (ion-exchange) chromatography” refers to a method that employs a chromatographic composition that uses an ion-exchange mode or mechanism of interaction. For example, proteins are amphoteric and bind to an ion-exchange material depending on the pH and ionic strength of the solution.

As used herein, “AEX (anion exchange) chromatography” refers to a method that employs a chromatographic composition wherein the fixed ion of the exchange material of the chromatographic composition carries a positive charge.

As used herein, “CEX (cation exchange) chromatography” refers to a method that employs a chromatographic composition wherein the fixed ion of the exchange material of the chromatographic composition carries a negative charge.

As used herein, “weak anion exchange chromatography” or “weak AEX chromatography” refers to a method wherein the anion exchange material of the chromatographic composition remains in a charged state in a narrow range of pH (e.g., 4-9). Diethyl aminoethyl (DEAE) is a non-limiting example of a weak anion exchange materials.

As used herein, a “strong chromatography composition” refers to an ion exchange (AEX or CEX) chromatography material (e.g., resin) that maintains its respective charge across a broad pH range, while “weak chromatography composition” refers to an ion exchange (AEX or CEX) chromatography material (e.g., resin) wherein the charge of the material varies with pH allowing for different selectivity and binding of solutes.

As used herein, “strong anion exchange chromatography” or “strong AEX chromatography” refers to a method wherein the anion exchange material of the chromatographic composition remains in a charged state over a wide range of pH (e.g., 2-12). Quaternary amine (QA) and Quaternized polyethyleneimine (XQ) are non-limiting examples of strong anion exchange materials.

As used herein, “strong cation exchange chromatography” or “strong CEX chromatography” refers to a method wherein the cation exchange material of the chromatographic composition remains in a charged state over a wide range of pH (e.g., 2-12). Sulfonic acid-based resins, and sulfopropyl or sulfoethyl resins are non-limiting examples of strong cation exchange materials.

As used herein, “Hydrophobic Interaction Chromatography (HIC)” refers to a method wherein the proteins bind and interact with the chromatographic composition based on their hydrophobicity. As used herein, “mixed-mode chromatography (MMC)” or “multimode chromatography” refers to method wherein the proteins bind and interact with the chromatographic composition through more than one interaction mode or mechanism (e.g., AEX and HIC).

As used herein, a “chromatography polishing step” refers to a chromatographic method employed to obtain higher purity product after a bulk separation from host cell proteins and media components has been performed. In certain embodiments, such bulk separation can be performed via an affinity chromatographic step.

As used herein “polishing” refers to subjecting the product of a bulk separation to chromatography methods, even if the bulk separation occurred using an initial chromatography method. A chromatography composition used to purify components from a bulk separation is a “polishing chromatography composition”. Polishing chromatography for AAVs generally separate empty, full and capsid charge variants among other impurities present in smaller quantities (for examples aggregates, DNA etc). The term "vector" refers to small carrier of nucleic acid molecule, a plasmid, virus (e.g., rAAV vector), or other vehicle that can be manipulated by insertion or incorporation of a nucleic acid. Vectors can be used for genetic manipulation (i.e., "cloning vectors"), to introduce/transfer polynucleotides into cells, and to transcribe or translate the inserted polynucleotide in cells. An "expression vector" is a vector that contains a gene or nucleic acid sequence with the necessary regulatory regions needed for expression in a host cell. A vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous nucleic acid sequence, expression control element (e.g., a promoter, enhancer), intron, inverted terminal repeats (ITRs), optional selectable marker, polyadenylation signal.

An rAAV vector is derived from adeno-associated virus. AAV vectors are useful as gene therapy vectors as they can introduce nucleic acid/genetic material into cells so that the nucleic acid/genetic material may be maintained in cells. Because AAV are not associated with pathogenic disease in humans, rAAV vectors are able to deliver heterologous nucleic acid sequences (e.g., therapeutic proteins and agents) to human patients without causing substantial AAV pathogenesis or disease.

The term "recombinant," as a modifier of vector, such as rAAV vectors, as well as a modifier of sequences such as recombinant polynucleotides and polypeptides, means that the compositions have been manipulated (i.e., engineered) in a fashion that generally does not occur in nature. A particular example of a recombinant AAV vector would be where a nucleic acid that is not normally present in the wild-type AAV genome is inserted within the viral genome. An example of would be where a nucleic acid (e.g., gene) encoding a therapeutic protein or polynucleotide sequence is cloned into a vector, with or without 5', 3' and/or intron regions that the gene is normally associated within the AAV genome. Although the term "recombinant" is not always used herein in reference to AAV vectors, as well as sequences such as polynucleotides, recombinant forms including AAV vectors, polynucleotides, etc., are expressly included in spite of any such omission.

A "rAAV vector" is derived from the wild type genome of a virus, such as AAV by using molecular methods to remove the wild type genome from AAV genome, and replacing with a nonnative (heterologous) nucleic acid, such as a nucleic acid encoding a therapeutic protein or polynucleotide sequence. Typically, for AAV one or both inverted terminal repeat (ITR) sequences of AAV genome are retained in the rAAV vector. A rAAV is distinguished from an AAV genome since all or a part of the AAV genome has been replaced with a non-native sequence with respect to the AAV genomic nucleic acid, such as with a heterologous nucleic acid encoding a therapeutic protein or poly-nucleotide sequence. Incorporation of a non-native sequence therefore defines the AAV as a "recombinant" AAV vector, which can be referred to as a "rAAV vector."

A recombinant AAV vector sequence can be packaged-referred to herein as a "particle" for subsequent infection (transduction) of a cell, ex vivo, in vitro or in vivo. Where a recombinant vector sequence is encapsidated or packaged into an AAV particle, the particle can also be referred to as a "rAAV" or "rAAV particle" or "rAAV virion." Such rAAV, rAAV particles and rAAV virions include proteins that encapsidate or package the vector genome. Particular examples include in the case of AAV, capsid proteins.

A vector "genome" refers to the portion of the recombinant plasmid sequence that is ultimately packaged or encapsidated to form a rAAV particle. In cases where recombinant plasmids are used to construct or manufacture recombinant AAV vectors, the AAV vector genome does not include the portion of the "plasmid" that does not correspond to the vector genome sequence of the recombinant plasmid. This non vector genome portion of the recombinant plasmid is referred to as the "plasmid backbone," which is important for cloning and amplification of the plasmid, a process that is needed for propagation and recombinant virus production, but is not itself packaged or encapsidated into rAAV particles. Thus, a vector "genome" refers to the nucleic acid that is packaged or encapsidated by rAAV.

"AAV helper functions" refer to AAV-derived coding sequences (proteins) which can be expressed to provide AAV gene products and AAV vectors that, in turn, function in trans for productive AAV replication and packaging. Thus, AAV helper functions include AAV open reading frames (ORFs), including rep and cap and others such as AAP for certain AAV serotypes. The Rep expression products have been shown to possess many functions, including, among others: recognition, binding and nicking of the AAV origin of DNA replication; DNA helicase activity; and modulation of transcription from AAV (or other heterologous) promoters. The Cap expression products (capsids) supply necessary packaging functions. AAV helper functions are used to complement AAV functions in trans that are missing from AAV vector genomes. An example of an AAV helper function is adenovirus E2 and/or E4, or virus associated RNA (VA RNA). Without being bound by theory, VA RNA is thought to provide helper function as a nucleic acid.

As used herein, the term “helper virus function(s)” or “helper function(s)” refers to function(s) enabled by nucleic acid sequences that encode proteins and/or nucleic acid molecules (e.g., RNA) that are relevant for AAV production. Such helper functions are encoded in a helper virus genome, which allow rAAV vector genome replication and packaging (in conjunction with Rep and Cap). AAV generally cannot be propagated by itself and therefore, to establish a productive viral infection, AAV is often coinfected with a helper virus. Adenovirus is believed to be the natural helper virus in the wild because clinical isolates of adenovirus are frequently contaminated with AAV, but herpesvirus and baculovirus also can supply complete helper activity in cell culture. Contemporary recombinant AAV (rAAV) virion production involves cotransfection of a host cell with an AAV vector plasmid and a construct which provides AAV helper functions to complement functions missing from the AAV vector plasmid. In this manner, the host cell is capable of AAV replication and packaging. The host cell is then infected with a helper virus to provide accessory functions. The helper virus is generally an infectious adenovirus (type 2 or 5), or herpesvirus. For adenovirus, helper functions have been identified as Ad Ela, E1B, E4, orf6, DBP, and viral associated RNA (VA RNA) (see Samulski et al., Annu. Rev. Virol. (2014), 1 :427- 451 and WO 1997/017458 incorporated herein by reference in their entirety).

An "AAV helper construct" refers generally to a nucleic acid sequence that includes nucleotide sequences providing AAV functions deleted from an AAV vector which is to be used to produce a transducing AVV vector for delivery of a nucleic acid sequence of interest, by way of gene therapy to a subject, for example. AAV helper constructs are commonly used to provide transient expression of AAV rep and/or cap genes to complement missing AAV functions that are necessary for AAV vector replication. Helper constructs generally lack AAV ITRs and can neither replicate nor package themselves. AAV helper constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. A number of AAV helper constructs have been described, such as plasmids pAAV/Ad and pIM29+45 which encode both Rep and Cap expression products (See, e.g., Samulski et al. (1989) J. Virol. 63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945). A number of other vectors have been described which encode Rep and/or Cap expression products (See, e.g., U.S. Pat. Nos. 5,139,941 and 6,376,237).

The term "accessory functions" refers to non-AAV derived viral and/or cellular functions upon which AAV is dependent for replication. The term includes proteins and RNAs that are required in AAV replication, including moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products and AAV capsid packaging. Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1) and vaccinia virus.

An "accessory function vector" refers generally to a nucleic acid molecule that includes polynucleotide sequences providing accessory functions. Such sequences can be on an accessory function vector, and transfected into a suitable host cell. The accessory function vector is capable of supporting rAAV virion production in the host cell. Accessory function vectors can be in the form of a plasmid, phage, transposon or cosmid. In addition, the full-complement of adenovirus genes are not required for accessory functions. For example, adenovirus mutants incapable of DNA replication and late gene synthesis have been reported to be permissive for AAV replication (Ito et al., (1970) J. Gen. Virol. 9:243; Ishibashi et al, (1971) Virology 45:317). Similarly, mutants within E2B and E3 regions have been shown to support AAV replication, indicating that the E2B and E3 regions are probably not involved in providing accessory functions (Carter et al., (1983) Virology 126:505). Adenoviruses defective in the El region, or having a deleted E4 region, are unable to support AAV replication. Thus, El A and E4 regions appear necessary for AAV replication, either directly or indirectly (Laughlin et al., (1982) J. Virol. 41 :868; Janik et al., (1981) Proc. Natl. Acad. Sci. USA 78: 1925; Carter et al., (1983) Virology 126:505). Other characterized Adenovirus mutants include: E1B (Laughlin et al. (1982), supra; Janik et al. (1981), supra; Ostrove et al., (1980) Virology 104:502); E2A (Handa et al., (1975) J. Gen. Virol. 29:239; Strauss et al., (1976) J. Virol. 17: 140; Myers et al., (1980) J. Virol. 35:665; Jay et al., (1981) Proc. Natl. Acad. Sci. USA 78:2927; Myers et al., (1981) J. Biol. Chem. 256:567); E2B (Carter, Adeno-Associated Virus Helper Functions, in I CRC Handbook of Parvoviruses (P. Tijssen ed., 1990)); E3 (Carter et al. (1983), supra); and E4 (Carter et al. (1983), supra; Carter (1995)). Studies of the accessory functions provided by adenoviruses having mutations in the E1B coding region have produced conflicting results, but ElB55k may be required for AAV virion production, while El B19k is not (Samulski et al., (1988) J. Virol. 62:206-210). In addition, International Publication WO 97/17458 and Matshushita et al., (1998) Gene Therapy 5:938-945, describe accessory function vectors encoding various Adenovirus genes. Exemplary accessory function vectors comprise an adenovirus VA RNA coding region, an adenovirus E4 ORF6 coding region, an adenovirus E2A 72 kD coding region, an adenovirus El A coding region, and an adenovirus E1B region lacking an intact ElB55k coding region. Such accessory function vectors are described, for example, in International Publication No. WO 01/83797.

As used herein, the term "serotype" is a distinction used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serologic distinctiveness is determined on the basis of the lack of cross-reactivity between antibodies to one AAV as compared to another AAV. Cross-reactivity differences are usually due to differences in capsid protein sequences/antigenic determinants (e.g., due to VP1, VP2, and/or VP3 sequence differences of AAV serotypes).

Under the traditional definition, a serotype means that the virus of interest has been tested against serum specific for all existing and characterized serotypes for neutralizing activity and no antibodies have been found that neutralize the virus of interest. As more naturally occurring virus isolates of are discovered and/or capsid mutants generated, there may or may not be serological differences with any of the currently existing serotypes. Thus, in cases where the new virus (e.g., AAV) has no serological difference, this new virus (e.g., AAV) would be a subgroup or variant of the corresponding serotype. In many cases, serology testing for neutralizing activity has yet to be performed on mutant viruses with capsid sequence modifications to determine if they are of another serotype according to the traditional definition of serotype. Accordingly, for the sake of convenience and to avoid repetition, the term "serotype" broadly refers to both serologically distinct viruses (e.g., AAV) as well as viruses (e.g., AAV) that are not serologically distinct that may be within a subgroup or a variant of a given serotype. rAAV vectors include any viral strain or serotype. As a non-limiting example, a rAAV plasmid or vector genome or particle (capsid) can be based upon any AAV serotype, such as AAV- 1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, for example. Such vectors can be based on the same of strain or serotype (or subgroup or variant), or be different from each other. As a non-limiting example, a rAAV plasmid or vector genome or particle (capsid) based upon one serotype genome can be identical to one or more of the capsid proteins that package the vector. In addition, a rAAV plasmid or vector genome can be based upon an AAV (e.g., AAV2) serotype genome distinct from one or more of the capsid proteins that package the vector genome, in which case at least one of the three capsid proteins could be, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hul4), AAV10, AAV11, AAV12, AAV13, Rh8, RhlO, Rh74, AAV3B, AAV-2i8, LK03, RHM4-1, DJ, DJ8, NP59, Anc-80 and variants thereof, including the variants of AAV capsids set forth in Pulicherla et al., Mol. Ther., 19(6) 1070-1078 (2011) (describing AAV9 variants including AAV9.47 among others), U.S. Patent No. 7,906,111 (describing AAV9(hul4) among others), U.S. Patent No. 10,532,111 (describing NP59 among others), U.S. Patent No. US10738087 (describing Anc-80 among others), W02012/145601, WO2013/158879, W02015/013313, WO2018/156654, US2013/0059732, U.S. Patent Nos. 9,169,299 (describing LK03), 9,840,719 (describing RHM4-1), 7,749,492, 7,588,772 (describing DJ and DJ8), and 9,587,282. rAAV vectors therefore include gene/protein sequences identical to gene/ protein sequences characteristic for a particular serotype, as well as mixed serotypes.

In various exemplary embodiments, a rAAV vector includes or consists of a capsid sequence at least 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) identical to one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hul4), AAV10, AAV11, AAV12, AAV13, Rh8, RhlO, Rh74, AAV3B, AAV-2i8, LK03, RHM4-1, DJ, DJ8, NP59, Anc-80 capsid proteins. In various exemplary embodiments, a rAAV vector includes or consists of a sequence at least 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) identical to one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hul4), AAV10, AAV11, AAV12, AAV13, Rh8, RhlO, Rh74, AAV3B, AAV-2i8, LK03, RHM4-1, DJ, DJ8, NP59, Anc-80 ITR(s). rAAV, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hul4), AAV10, AAV11, AAV12, AAV13, Rh8, RhlO, Rh74, AAV3B, AAV- 2i8, LK03, RHM4-1, DJ, DJ8, NP59, Anc-80 and variant, hybrid and chimeric sequences, can be constructed using recombinant techniques that are known to the skilled artisan, to include one or more heterologous polynucleotide sequences (trans-genes) flanked with one or more functional AAV ITR sequences. Such vectors have one or more of the wild type AAV genes deleted in whole or in part, but retain at least one functional flanking ITR sequence(s), as necessary for the rescue, replication, and packaging of the recombinant vector into a rAAV vector particle. A rAAV vector genome would therefore include sequences required in cis for replication and packaging (e.g., functional ITR sequences).

The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to all forms of nucleic acid, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids include genomic DNA, cDNA and antisense DNA, and spliced or unspliced mRNA, rRNA tRNA and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), small or short interfering (si)RNA, trans-splicing RNA, or antisense RNA). Nucleic acids include naturally occurring, synthetic, and intentionally modified or altered polynucleotides. Nucleic acids can be single, double, or triplex, linear or circular, and can be of any length. In discussing nucleic acids, a sequence or structure of a particular polynucleotide may be described herein according to the convention of providing the sequence in the 5' to 3' direction.

A "heterologous" nucleic acid sequence refers to a polynucleotide inserted into a AAV plasmid or vector for purposes of vector mediated transfer/delivery of the polynucleotide into a cell. Heterologous nucleic acid sequences are distinct from AAV nucleic acid, i.e., are non-native with respect to AAV nucleic acid. Once transferred/delivered into the cell, a heterologous nucleic acid sequence, contained within the vector, can be expressed (e.g., transcribed, and translated if appropriate). Alternatively, a transferred/delivered heterologous polynucleotide in a cell, contained within the vector, need not be expressed. Although the term "heterologous" is not always used herein in reference to nucleic acid sequences and polynucleotides, reference to a nucleic acid sequence or polynucleotide even in the absence of the modifier "heterologous" is intended to include heterologous nucleic acid sequences and polynucleotides in spite of the omission.

The "polypeptides," "proteins" and "peptides" encoded by the "nucleic acid sequence," include full-length native sequences, as with naturally occurring proteins, as well as functional subsequences, modified forms or sequence variants so long as the subsequence, modified form or variant retains some degree of functionality of the native full-length protein. Such polypeptides, proteins and peptides encoded by the nucleic acid sequences can be but are not required to be identical to the endogenous protein that is defective, or whose expression is insufficient, or deficient in the treated mammal.

A "transgene" is used herein to conveniently refer to a nucleic acid (e.g., heterologous) that is intended or has been introduced into a cell or organism. Transgenes include any nucleic acid, such as a heterologous nucleic acid encoding a therapeutic protein or polynucleotide sequence.

In a cell having a transgene, the transgene has been introduced/transferred by way of a plasmid or a AAV vector, "transduction" or "transfection" of the cell. The terms "transduce" and "transfect" refer to introduction of a molecule such as a nucleic acid into a host cell (e.g., HEK293) or cells of an organism. The transgene may or may not be integrated into genomic nucleic acid of the recipient cell. If an introduced nucleic acid becomes integrated into the nucleic acid (genomic DNA) of the recipient cell or organism it can be stably maintained in that cell or organism and further passed on to or inherited by progeny cells or organisms of the recipient cell or cells of an organism.

A "host cell" denotes, for example, microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of an AAV vector plasmid, AAV helper construct, an accessory function vector, or other transfer DNA. The term includes the progeny of the original cell which has been transfected. Thus, a "host cell" generally refers to a cell which has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. Exemplary host cells include human embryonic kidney (HEK) cells such as HEK293.

A "transduced cell" is a cell into which a transgene has been introduced. Accordingly, a "transduced" cell means a genetic change in a cell following incorporation of an exogenous molecule, for example, a nucleic acid (e.g., a transgene) into the cell. Thus, a "transduced" cell is a cell into which, or a progeny thereof in which an exogenous nucleic acid has been introduced. The cell(s) can be propagated (cultured) and the introduced protein expressed or nucleic acid transcribed, or vector, such as rAAV, produced by the cell. For gene therapy uses and methods, a transduced cell can be in a subject.

As used herein, the term "stable" in reference to a cell, or "stably integrated" means that nucleic acid sequences, such as a selectable marker or heterologous nucleic acid sequence, or plasmid or vector has been inserted into a chromosome (e.g., by homologous recombination, non- homologous end joining, transfection, etc.) or is maintained in the recipient cell or host organism extrachromosomally, and has remained in the chromosome or is maintained extrachromosomally for a period of time. In the case of culture cells, nucleic acid sequences, such as a heterologous nucleic acid sequence, or plasmid or vector has been inserted into a chromosome can be maintained over the course of a plurality of cell passages.

A "cell line" refers to a population of cells capable of continuous or prolonged growth and division in vitro under appropriate culture conditions. Cell lines can, but need not be, clonal populations derived from a single progenitor cell. In cell lines, spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations, as well as during prolonged passaging in tissue culture. Thus, progeny cells derived from the cell line may not be precisely identical to the ancestral cells or cultures. An exemplary cell line applicable to the purification methods disclosed herein is HEK293.

An "expression control element" refers to nucleic acid sequence(s) that influence expression of an operably linked nucleic acid. Control elements, including expression control elements as set forth herein such as promoters and enhancers. rAAV vectors can include one or more "expression control elements." Typically, such elements are included to facilitate proper heterologous polynucleotide transcription and if appropriate translation (e.g., a promoter, enhancer, splicing signal for introns, maintenance of the correct reading frame of the gene to permit in-frame translation of mRNA and, stop codons etc.). Such elements typically act in cis, referred to as a "cis acting" element, but may also act in trans.

Expression control can be exerted at the level of transcription, translation, splicing, message stability, etc. Typically, an expression control element that modulates transcription is juxtaposed near the 5' end (i.e., "upstream") of a transcribed nucleic acid. Expression control elements can also be located at the 3' end (i.e., "downstream") of the transcribed sequence or within the transcript (e.g., in an intron). Expression control elements can be located adjacent to or at a distance away from the transcribed sequence (e.g., 1-10, 10-25, 25-50, 50-100, 100 to 500, or more nucleotides from the polynucleotide), even at considerable distances. Nevertheless, owing to the length limitations of rAAV vectors, expression control elements will typically be within 1 to 1000 nucleotides from the transcribed nucleic acid.

Functionally, expression of operably linked nucleic acid is at least in part controllable by the element (e.g., promoter) such that the element modulates transcription of the nucleic acid and, as appropriate, translation of the transcript. A specific example of an expression control element is a promoter, which is usually located 5' of the transcribed sequence. A promoter typically increases an amount expressed from operably linked nucleic acid as compared to an amount expressed when no promoter exists.

An "enhancer" as used herein can refer to a sequence that is located adjacent to the nucleic acid sequence, such as selectable marker, or heterologous nucleic acid sequence Enhancer elements are typically located upstream of a promoter element but also function and can be located downstream of or within a sequence. Hence, an enhancer element can be located upstream or downstream, e.g., within 100 base pairs, 200 base pairs, or 300 or more base pairs of the as selectable marker, and/or a heterologous nucleic acid encoding a therapeutic protein or polynucleotide sequence. Enhancer elements typically increase expression of an operably linked nucleic acid above expression afforded by a promoter element.

The term "operably linked" means that the regulatory sequences necessary for expression of a nucleic acid sequence are placed in the appropriate positions relative to the sequence so as to effect expression of the nucleic acid sequence. This same definition is sometimes applied to the arrangement of nucleic acid sequences and transcription control elements (e.g. promoters, enhancers, and termination elements) in an expression vector, e.g., rAAV vector.

In the example of an expression control element in operable linkage with a nucleic acid, the relationship is such that the control element modulates expression of the nucleic acid. More specifically, for example, two DNA sequences operably linked means that the two DNAs are arranged (cis or trans) in such a relationship that at least one of the DNA sequences is able to exert a physiological effect upon the other sequence.

Accordingly, additional elements for vectors include, without limitation, an expression control (e.g., promoter/enhancer) element, a transcription termination signal or stop codon, 5' or 3' untranslated regions (e.g., poly-adenylation (poly A) sequences) which flank a sequence, such as one or more copies of an AAV ITR sequence, or an intron.

Further elements include, for example, filler or stuffer polynucleotide sequences, for example to improve packaging and reduce the presence of contaminating nucleic acid. AAV vectors typically accept inserts of DNA having a size range which is generally about 4 kb to about 5.2 kb, or slightly more. Thus, for shorter sequences, inclusion of a stuffer or filler in order to adjust the length to near or at the normal size of the virus genomic sequence acceptable for vector packaging into a rAAV particle. In various embodiments, a filler/ stuff er nucleic acid sequence is an untranslated (non-protein encoding) segment of nucleic acid. For a nucleic acid sequence less than 4.7 Kb, the filler or stuffer polynucleotide sequence has a length that when combined (e.g., inserted into a vector) with the sequence has a total length between about 3.0-5.5 Kb, or between about 4.0-5.0 Kb, or between about 4.3-4.8 Kb.

A "therapeutic protein" in one embodiment is a peptide or protein that may alleviate or reduce symptoms that result from an insufficient amount, absence or defect in a protein in a cell or subject. A "therapeutic" protein encoded by a transgene can confer a benefit to a subject, e.g., to correct a genetic defect, to correct a gene (expression or functional) deficiency, etc.

Non-limiting examples of heterologous nucleic acids encoding gene products (e.g., therapeutic proteins) which are useful in accordance with the subject matter disclosed herein include those that may be used in the treatment of a disease or disorder including, but not limited to, "hemostasis" or blood clotting disorders such as hemophilia A, hemophilia A patients with inhibitory antibodies, hemophilia B, deficiencies in coagulation Factors, VII, VIII, IX and X, XI, V, XII, II, von Willebrand factor, combined FV/FVIII deficiency, thalassemia, vitamin K epoxide reductase Cl deficiency, gamma-carboxylase deficiency; anemia, bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC); over-anticoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, small molecule antithrombotics (i.e. FXa inhibitors); and platelet disorders such as, Bernard Soulier syndrome, Glanzman thromblastemia, and storage pool deficiency.

Nucleic acid molecules, vectors such as cloning, expression vectors (e.g., vector genomes) and plasmids, may be prepared using recombinant DNA technology methods. The availability of nucleotide sequence information enables preparation of nucleic acid molecules by a variety of means. For example, a heterologous nucleic acid encoding Factor IX (FIX) comprising a vector or plasmid can be made using various standard cloning, recombinant DNA technology, via cell expression or in vitro translation and chemical synthesis techniques. Purity of polynucleotides can be determined through sequencing, gel electrophoresis and the like. For example, nucleic acids can be isolated using hybridization or computer-based database screening techniques. Such techniques include, but are not limited to: (1) hybridization of genomic DNA or cDNA libraries with probes to detect homologous nucleotide sequences; (2) antibody screening to detect polypeptides having shared structural features, for example, using an expression library; (3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of annealing to a nucleic acid sequence of interest; (4) computer searches of sequence databases for related sequences; and (5) differential screening of a subtracted nucleic acid library.

The term "isolated," when used as a modifier of a composition, means that the compositions are made by the hand of man or are separated, completely or at least in part, from their naturally occurring in vivo environment. Generally, isolated compositions are substantially free of one or more materials with which they normally associate with in nature, for example, one or more protein, nucleic acid, lipid, carbohydrate, cell membrane.

With respect to protein, the term "isolated protein" or "isolated and purified protein" is sometimes used herein. This term refers primarily to a protein produced by expression of a nucleic acid molecule. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in "substantially pure" form.

The term "isolated" does not exclude combinations produced by the hand of man, for example, a rAAV and a pharmaceutical formulation. The term "isolated" also does not exclude alternative physical forms of the composition, such as hybrids/chimeras, multimers/oligomers, modifications (e.g., phosphorylation, glycosylation, lipidation) or derivatized forms, or forms expressed in host cells produced by the hand of man.

The term "substantially pure" refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). The preparation can comprise at least 75% by weight, or about 90-99% by weight, of the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g., chromatographic methods, agarose or poly-acrylamide gel electrophoresis, HPLC analysis, and the like).

The phrase "consisting essentially of when referring to a particular nucleotide sequence or amino acid sequence means a sequence having the properties of a given sequence. For example, when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the basic and novel characteristics of the sequence.

2. Purification Methods

In certain embodiments, the methods of the present disclosure comprise: (a) contacting a sample comprising rAAV vector particles to an AAV capture composition, e.g. an affinity chromatography composition, to produce a capture step eluate comprising rAAV vector particles; (b) contacting said capture step eluate to a first polishing chromatography composition to produce a first polishing eluate comprising rAAV vector particles; and (c) contacting said first polishing eluate to a second polishing chromatography composition to produce a second polishing eluate comprising differentially separated rAAV vector capsid variants.

In certain embodiments, the first polishing chromatography composition and the second polishing chromatography composition are selected from distinct separation strategies, e.g., strong AEX chromatography; strong CEX chromatography; HIC; MMC; and weak AEX chromatography. In certain embodiments, the first polishing chromatography composition and the second polishing chromatography composition are selected from the same separation strategy, (e.g., both compositions are strong AEX chromatography compositions) but comprise distinct ligands (e.g., distinct fixed ions in the case of IEX chromatography compositions).

In certain embodiments, the first polishing chromatography composition is selected from the group consisting of: a strong AEX chromatography composition; a strong CEX chromatography composition; a HIC composition; a MMC composition; and a weak AEX chromatography composition; while the second polishing chromatography composition is selected from the group consisting of: a strong AEX chromatography composition; a strong CEX chromatography composition; a HIC composition; a MMC composition; and a weak AEX chromatography composition. Non-limiting examples of chromatographic methods that find use in the methods described herein are known in the art, e.g., Saraswat et al. (2013) Biomed Res Int. 2013:312709; and Zhang et al. (2016) Journal of Pharmaceutical and Biomedical Analysis, Volume 128:73-88., the contents of which are incorporated herein by reference in their entirety.

2.1 Affinity Chromatography

Affinity chromatographic compositions are typically composed of a ligand linked or conjugated to a substrate. Particular examples of ligands include AAV binding antibodies. Such substrates include sepharose and other materials typically used in such affinity purification applications and can be made or are commercially available (e.g., AVB Sepharose™ High Performance, GE Healthcare, Marlborough, Mass.). In certain embodiments of the presently disclosed subject matter, an AAV affinity chromatographic composition can comprise a protein or ligand that binds to AAV capsid protein. Non-limiting examples of a protein include an antibody that binds to AAV capsid protein. More specific non-limiting examples include a single-chain Llama antibody (Camelid) that binds to AAV capsid protein.

Equilibration buffers and solutions for washes and elutions for affinity chromatographic compositions can be Tris or acetate based. For example, affinity chromatography compositions can be equilibrated with a Tris buffer, e.g., at about 1 mM to about 5 mM, about 5 mM to about 50 mM, or 5 mM to about 20 mM, and with NaCl at about 50 mM to about 100 mM, about 100 mM to about 150 mM, about 150 mM to about 200 mM, about 200 mM to about 250 mM, about 250 mM to about 300 mM, or any amount at or within these stated ranges.

Typical equilibration buffers for use in affinity chromatography can comprise a pH of from about pH 7.5 to about pH 9.0, more typically from about pH 8.0 to about pH 8.5, and even more typically a pH such as pH 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5.

After equilibration, a sample can then be contacted to the affinity chromatographic composition. Subsequently, the rAAV particles can be eluted from the affinity chromatographic composition by reducing pH of the buffer to less than 7.0. Elution buffers can be acetate based and typically pH is less than 5.0, more typically less than 4.0, such as less than 3.0, more specifically between about 2.0 and 3.0, or any pH at or within these stated ranges.

In certain embodiments, the volumes of buffer for equilibration, washing and elution can be based upon the amount of chromatography media and/or column size to achieve rAAV particle recovery. Typical volumes are 1-10 column volumes.

Affinity chromatography eluate can be collected following the elution(s)/flow through from each of the affinity chromatography steps. AAV can be detected in the fractions using standard techniques, such as monitoring UV absorption at 260 and 280.

2.2 Ion-Exchange Chromatography (IEX)

Proteins are amphoteric molecules, and any protein will bind to an ion exchange resin depending on the pH and ionic strength of the solution. IEX provides high resolution under mild conditions with high binding capacity. The pH and ionic strength of the buffer selected for binding and elution affect the separation process of different products on IEX. Theoretically, any salt can be used for elution because they all modulate electrostatic interactions, thus binding and elution.

The use of cation or anion exchange chromatography media, the nature of the media used

(i.e., strong or weak ion exchangers) and conditions of salt concentration, buffer used, and pH, can vary based upon the rAAV capsid (i.e., rAAV capsid serotype or pseudotype). While rAAV capsid structure typically share features such as size and shape, capsids may have different amino acid sequences that result in subtle differences of molecular topology and surface charge distribution. Thus, capsid sequence variants are amenable to purification by the methods disclosed herein.

2.2.1 Anion Exchange Chromatography (AEX)

An ion exchanger is classified as an anion-exchange material when the fixed ion carries a positive charge. Anion exchange chromatography functions to separate AAV particles from proteins, cellular and other components present in the clarified lysate and/or column eluate from the size exclusion chromatography. Anion exchange chromatography can also be used to control the amount of AAV empty capsids in the eluate. For example, an anion exchange chromatographic composition comprising rAAV vector bound thereto can be washed with NaCl at a modest concentration (e.g., about 100 mM to about 125 mM, such as about 110 mM to about 115 mM) and a portion of the empty capsids can be eluted in the flow-through without substantial elution of the rAAV vectors. Subsequently, rAAV vector bound to the anion exchange chromatographic composition can be eluted using NaCl at a higher concentration (e.g., about 130-300 mM NaCl), thereby producing a polishing chromatographic eluate with reduced or depleted amounts of AAV empty capsids and proportionally increased amounts of rAAV.

Exemplary anion exchange resins include, without limitation, those based on polyamine resins and other resins. Examples of strong anion exchange resins include those based generally on the quaternized nitrogen atom including, without limitation, quaternary ammonium salt resins such as trialkylbenzyl ammonium resins. Suitable exchange chromatography include without limitation, MACRO PREP Q (strong anion-exchanger available from BioRad, Hercules, Calif.); UNOSPHERE Q (strong anion-exchanger available from BioRad, Hercules, Calif.); POROS 50HQ (strong anion-exchanger available from Applied Biosystems, Foster City, Calif.); POROS XQ (strong anion-exchanger available from Applied Biosystems, Foster City, Calif.); POROS 50D (weak anion-exchanger available from Applied Biosystems, Foster City, Calif.); POROS 50PI (weak anion-exchanger available from Applied Biosystems, Foster City, Calif.); Capto Q, Capto XQ, Capto Q ImpRes, and SOURCE 30Q (strong anion-exchanger available from GE healthcare, Marlborough, Mass.); DEAE SEPHAROSE (weak anion-exchanger available from Amersham Biosciences, Piscat-away, N.J.); Q SEPHAROSE (strong anion-exchanger available from Amersham Biosciences, Piscataway, N.J.); QA (strong anion-exchanger available from Sartorius, Ajdovscina, Slovenia); QA HR (strong anion-exchanger available from Sartorius, Ajdovscina, Slovenia). Additional exemplary anion exchange resins include aminoethyl (AE), diethylaminoethyl (DEAE), diethylaminopro-pyl (DEPE) and quaternary amino ethyl (QAE). Exemplary anion exchange membranes include, without limitation, Sartobind Q (strong ani on-exchanger available from Sartorius, Ajdovscina, Slovenia); Mustang Q (strong ani on- exchanger available from Pall, Port Washington, NY).

Typical equilibration buffers and solutions for washes and elutions for anion exchange chromatography an appropriate at a pH of from about pH 7.5 to about pH 12, more typically from about pH 8.0 to about pH 10, and even more typically from about pH 8.0 to about pH 9.0, such as pH 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0.

Appropriate equilibration buffers and solutions for washes and elutions for anion exchange chromatographic compositions are generally cationic or zwitterionic in nature. Such buffers include, without limitation, buffers with the following buffer agents: N-methylpiperazine; piperazine; Bis-Tris; Bis-Tris propane; Triethanolamine; Tris; N-methyl di ethanolamine; 1,3 - diaminopropane; ethanolamine; acetic acid, and the like. To elute the sample, the ionic strength of the starting buffer is increased using a salt, such as NaCl, KC1, sulfate, formate or acetate. Such equilibration buffers and solutions for washes and elutions can have the foregoing buffering agents from about 5 mM to about 100 mM, more typically from about 10 mM to about 50 mM.

In one embodiment, the anion exchange chromatography composition is first equilibrated, sample contacted, and washed with a low salt concentration, e.g., about 10 mM to about 150 mM of NaCl, such as 10-30, 30-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-100 mM, or any concentration at or within these ranges. Following a first wash, the chromatography composition can be treated with a higher salt concentration in order to elute impurities such as AAV empty capsids, such as a higher NaCl concentration, or with another buffer with a greater ionic strength. One example for use as the second buffer is a Tris-based buffer with a NaCl concentration of about 110 mM-125 mM, or any concentration at or within these stated ranges.

After additional impurities are eluted from the chromatographic composition, the AAV particles can, in certain embodiments, be recovered by elution with a higher concentration of salt. Exemplary elution buffers can include Tris-based buffers with NaCl concentrations of 125 mM or greater, such as about 125 mM to about 150 mM, about 150 mM to about 200 mM or about 200 mM to about 250 mM NaCl, or any concentration at or within these stated ranges.

In certain embodiments, polyethylene glycol (PEG) can be included in the anion exchange chromatography composition wash solution. This is referred to as polyethylene glycol (PEG) modulated chromatography. PEG wash solutions can be applied to the anion exchange chromatography composition prior to elution of AAV vector particles. In certain embodiments, the PEG in such wash solutions has an average molecular weight in a range of about 1,000 to about 80,000 g/mol, inclusive. In certain embodiments, the amounts of PEG in such wash solutions range from about 0.1% to about 20% PEG or any amount at or within these stated ranges, or from about 1% to about 10% PEG or any amount at or within these stated ranges.

2.2.2 Cation Exchange Chromatography (CEX)

The ion exchange is classified as a cation-exchange chromatographic composition when the fixed ion carries a negative charge. Examples of strong cation exchange resins capable of binding rAAV particles over a wide pH range include, without limitation, any sulfonic acid-based resins as indicated by the presence of the sulfonate functional group, including aryl and alkyl substituted sulfonates, such as sulfopropyl or sulfoethyl resins. Representative compositions include, but are not limited to, POROS HS, POROS HS 50, POROS XS, POROS SP, and POROS S (strong cation exchangers available from Thermo Fisher Scientific, Inc., Waltham, Mass.). Additional examples include Capto S, Capto S ImpAct, Capto S ImpRes (strong cation exchangers available from GE Healthcare, Marlborough, Mass.), and commercial DOWEX®, AMBERLITE®, and AMBERLYST® families of resins available from Aldrich Chemical Company (Mil-liwaukee, Wis.). Weak cation exchange compositions include, without limitation, any carboxylic acid based resins. Exemplary cation exchange compositions also include carboxymethyl (CM), phospho (based on the phosphate functional group), methyl sulfonate (S) and sulfopropyl (SP) resins.

Chromatography medium such as cation exchange can be equilibrated, washed and eluted with various buffers under various conditions such as pH, and buffer volumes. The following is intended to describe particular non-limiting examples.

Cation exchange chromatography may be equilibrated using standard buffers and according to the manufacturer's specifications. For example, chromatography media can be equilibrated with a phosphate buffer, at about 5 mM to about 100 mM, or about 10 mM to about 50 mM, such as 10 mM to about 30 mM, and sodium chloride. After equilibration, sample is then loaded. Subsequently, the chromatography media is washed at least once, or more, e.g., 2-10 times. Elution from the chromatography media is by way of a high salt buffer, at least once, but elution may be 2 or more times with the same or a higher salt buffer.

Typical equilibration buffers and solutions for washes and elutions for cation exchange chromatography are at an appropriate pH, of from about pH 3 to about pH 8, more typically from about pH 4 to about pH 7.5, such as pH 6.0-6.5, 6.5-7.0, 7.0-7.5, or any pH at or between the stated ranges such as, 7.0, 7.1, 7.2, 7.3 or 7.4.

Appropriate equilibration buffers and solutions for washes and elutions for cation exchange columns are known in the art and are generally anionic. Such buffers include, without limitation, buffers with the following buffer ions: phosphate, acetate, citrate, borate, or sulfate. In one embodiment, the cation exchange chromatography media is first equilibrated, sample applied, and washed with a low salt concentration, e.g., about 10 mM to about 150 mM of NaCl, such as 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 60-125 mM, or any concentration at or within these ranges, such as, 100 mM.

Following a first wash, the chromatography composition may be treated with a higher salt concentration in order to elute impurities, such as a higher NaCl concentration, or with another buffer with a greater ionic strength. After additional impurities are eluted from the column, to elute rAAV particles, the ionic strength of the buffer may be increased using a salt, such as NaCl, KC1, sulfate, formate or acetate, and recovered. In one embodiment, elution is with a high salt concentration, e.g., about 200 mM to about 500 mM of NaCl, or any concentration at or within these ranges, such as 250 mM, 300 mM, 350 mM, or 400 mM.

Additional components can be included in the equilibration buffers and solutions for washes and elutions. For example, a wash buffer for cation exchange chromatography can include an anionic surfactant such as sarkosyl (e.g., about 1 mM to about 10 mM), a wash buffer for anion exchange chromatography can include a cationic surfactant such as Dodecylt-rimethylammonium chloride (e.g., about 1 mM to about 10 mM).

2.3 Hydrophobic Interaction Chromatography (HIC)

Proteins bind to HIC hydrophobic ligands on resins based on their hydrophobicity, indicating the separation conditions such as retention times of a given protein and, therefore, performance of HIC. Protein hydrophobicity is determined by the amino acids that constitute the proteins, with high concentrations of salt (e.g., ammonium sulphate) exposing the surface hydrophobic patches and thus enabling the binding of proteins to the HIC composition. A decreasing gradient of salts performs subsequent elution. Representative matrices include but are not limited to Capto Butyl ImpRes (hydrophobic interaction resin available from Millipore Sigma, Darmstadt, Germany); Capto Phenyl ImpRes (hydrophobic interaction resin available from Millipore Sigma, Darmstadt, Germany).

2.4. Mixed-Mode Chromatography (MMC)

Mixed-mode chromatography (MMC) or multimode chromatography is a chromatographic method in which solutes interact with the stationary phase through more than one interaction mode or mechanism (e.g., AEX and HIC). MMC has been used as an alternative or complementary tool to traditional reversed-phased (RP), ion exchange (IEX) and normal phase chromatography (NP). Unlike RP, NP and IEX chromatography, in which hydrophobic interaction, hydrophilic interaction and ionic interaction respectively are the dominant interaction modes, mixed-mode chromatography employs a combination of two or more of these interaction modes. Representative matrices include but are not limited to Capto Adhere ImpRes (multimodal anion exchanger available from Millipore Sigma, Darmstadt, Germany); PrimaS (multimodal chromatography ligand that combines elements of hydrogen bonding with anion exchange chromatography available from Sartorius, Ajdovscina, Slovenia). Capto Core 400 (multimodal ligand with dual functionality of size exclusion and binding chromatography available from Cytiva).

3. Production of rAA V

Methods to produce rAAV virions are known in the art and any such methods can be adapted to prepare samples for the differential separation methods disclosed herein. For example, transfection using rAAV vector and AAV helper sequences in conjunction with coinfection with one AAV helper viruses (e.g., adenovirus, herpesvirus, or vaccinia virus) or transfection with a rAAV vector, an AAV helper vector, and an accessory function vector. Non-limiting methods for generating rAAV virions are described, for example, in U.S. Pat. Nos. 6,001,650 and 6,004,797, International Application PCT/US16/64414 (published as WO 2017/096039) and U.S. Provisional Application Nos. 62/516,432 and 62/531,626. Following recombinant rAAV vector production (i.e., vector generation in cell culture systems), rAAV virions can be obtained from the host cells and cell culture supernatant and purified as set forth herein as well as using strategies known in the art.

As an initial step, in certain embodiments, host cells producing rAAV virions can be harvested, optionally in combination with harvesting cell culture supernatant (medium) in which the host cells (suspension or adherent) producing rAAV virions have been cultured. In certain embodiments, the harvested cells, and optionally the cell culture supernatant, can be used as is, as appropriate, or concentrated. In certain embodiments, e.g., if infection has been employed to express accessory functions, residual helper virus can be inactivated. For example, adeno-virus can be inactivated by heating to temperatures of approximately 60° C for, e.g., 20 minutes or more, which inactivates only the helper virus since AAV is heat stable while the helper adenovirus is not.

In certain embodiments, cells and/or supernatant of the harvest can be lysed by disrupting the cells, for example, by chemical or physical means, such as detergent, microfluidization and/or homogenization, to release the rAAV particles. Concurrently during cell lysis or subsequently after cell lysis, a nuclease such as benzonase can be added to degrade contaminating DNA. In certain embodiments, the resulting lysate can be clarified to remove cell debris, such as by filtering and/or centrifuging, to render a clarified cell lysate. In certain embodiments, the lysate can be filtered with a micron diameter pore size filter (such as a 0.1-10.0 pm pore size filter, for example, a 0.45 pm and/or pore size 0.2 pm filter), to produce a clarified lysate. In certain embodiments, the lysate (optionally clarified) can contain rAAV particles (including bona fide rAAV vectors, AAV empty capsids, as well as other rAAV capsid variants) and rAAV vector production/process related impurities, such as soluble cellular components from the host cells that can include, inter alia, cellular proteins, lipids, and/or nucleic acids, and cell culture medium components. The optionally clarified lysate can then be subjected to additional purification steps to purify rAAV particles (including bona fide rAAV vectors) from impurities as well as to differentially separate rAAV capsid variants using chromatography. Clarified lysate can, in certain embodiments, be diluted or concentrated with an appropriate buffer prior to the first step of chromatography.

As disclosed herein, a sample comprising rAAV vector particles can be produced by cell lysis, optional clarifying, and optional dilution and/or concentration. In certain embodiments, a plurality of sequential chromatography steps can be used to purify rAAV particles. Such methods typically, but not necessarily, exclude a step of cesium chloride gradient ultracentrifugation.

A non-limiting example of ultrafiltration/diafiltration is tangential flow filtration (TFF). For example, a hollow fiber membrane with a nominal pore size corresponding to a 100 kDa molecular weight cutoff can be employed. In certain embodiments, such strategies art employed so that large amounts of AAV vector can be prepared even when present in larger volumes of eluate.

Eluates comprising rAAV particles from any of the affinity chromatography, ion-exchange chromatography, anion exchange chromatography, cation exchange, hydrophobic interaction chromatography, and/or mixed-mode chromatography steps as described herein can, if desired, be efficiently concentrated by ultrafiltration/diafiltration. Reduction in volume can be controlled by the skilled artisan. In particular non-limiting examples the reduction in volume achieved is between abut 1-30 fold , inclusive. Thus, a 1-fold reduction reduces the volume by half, e.g., 1000 ml is concentrated to 500 mL. A 10 fold reduction reduces the volume by a factor of 10, e.g., 2000 ml is concentrated to 200 mL. A 20 fold reduction reduces the volume by a factor of 20, e.g., 2000 ml is concentrated to 100 mL. A 30 fold reduction reduces the volume by a factor of 30, e.g., 2000 ml is concentrated to 66.67 mL.

The cell lysate and chromatography eluates comprising rAAV particles from any of the affinity chromatography, ion-exchange chromatography, anion exchange chromatography, cation exchange, hydrophobic interaction chromatography, and/or mixed-mode chromatography steps as described herein can, if desired, be diluted. Typical dilutions range from 25-100%, 1-2 fold, 2-5 fold or any volume or amount at or within these stated ranges. In certain embodiments of the presently disclosed subject matter, the methods disclosed herein achieve substantial recovery of rAAV particles. For example, methods of the present disclosure can achieve recovery of approximately 40-70% of the total rAAV vector particles from the host cells and host cell culture supernatant harvested. In certain embodiments, rAAV particles are present in the final (e.g., second polishing chromatography step) eluate at a concentration of about 100 mg/mL. In certain embodiments, the rAAV vector particles can be present in the final (e.g., second polishing chromatography step) eluate at a concentration of about 10¹ °’l 0¹¹ particles per mL, 10ⁿ-10¹² particles per mL, 10¹²-l 0¹³ particles per mL, or more.

Alternatively, if rAAV vector particle concentrations are less, purified rAAV particles can be concentrated. For example, purified AAV particles can be concentrated by ultrafiltration/diafiltration (e.g., TFF). If higher concentrations of vector are desired, purified AAV particles can be concentrated to 10¹²-l 0¹³ particles per mL, or more, 10¹³- 10¹⁴ particles per mL or more, by ultrafiltration/diafiltration (e.g., TFF), or even higher.

In other embodiments, rAAV particles with packaged genomes (i.e., bona fide rAAV vector particles) are "substantially free of "AAV-encapsidated nucleic acid impurities" when at least about 30% or more of the virions present are rAAV particles with packaged genomes (i.e., bona fide rAAV vector particles). Production of rAAV particles with packaged genomes (i.e., bona fide rAAV vector particles) substantially free of AAV-encapsidated nucleic acid impurities can be from about 40% to about 20% or less, about 20% to about 10%, or less, about 10% to about 5% or less, about 5% to about 1% or less than 1% or less of the product comprises AAV-encapsidated nucleic acid impurities.

Methods to determine infectious titer of AAV vector containing a transgene are known in the art (See, e.g., Zhen et al., (2004) Hum. Gene Ther. (2004) 15:709). Methods for assaying for empty capsids and AAV vector particles with packaged genomes are known (See, e.g., Grimm et al., Gene Therapy (1999) 6: 1322-1330; Sommer et al., Molec. Ther. (2003) 7: 122-128).

To determine the presence or amount of degraded/denatured capsid, purified AAV can be subjected to SDS-polyacrylamide gel electrophoresis, consisting of any gel capable of separating the three capsid proteins, for example, a gradient gel, then running the gel until sample is separated, and blotting the gel onto nylon or nitrocellulose membranes. Anti-AAV capsid antibodies are then used as primary antibodies that bind to denatured capsid proteins (See, e.g., Wobus et al., J. Virol. (2000) 74:9281-9293). A secondary antibody that binds to the primary antibody contains a means for detecting the primary antibody. Binding between the primary and secondary antibodies can be detected semi-quantitatively to determine the amount of capsids. Another method would be analytical HPLC with a SEC column or analytical ultracentrifuge. In certain embodiments of the presently disclosed subject matter, rAAV vector particles are derived from an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hul4), AAV10, AAV11, AAV12, AAV13, Rh8, RhlO, Rh74, AAV3B, AAV-2i8, LK03, RHM4-1, DJ, DJ8, NP59, Anc-80.

In certain embodiments of the presently disclosed subject matter, rAAV vector particles comprise a capsid sequence having 70% or more identity to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hul4), AAV10, AAV11, AAV12, AAV13, Rh8, RhlO, Rh74, AAV3B, AAV-2i8, LK03, RHM4-1, DJ, DJ8, NP59, Anc-80.

In certain embodiments of the presently disclosed subject matter, rAAV vector particles comprise an ITR sequence having 70% or more identity to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hul4), AAV10, AAV11, AAV12, AAV13, Rh8, RhlO, Rh74, AAV3B, AAV-2i8, LK03, RHM4-1, DJ, DJ8, NP59, Anc-80 ITR sequence.

In certain embodiments of the presently disclosed subject matter, cells are suspension or adherent cells.

In certain embodiments of the presently disclosed subject matter, cells are mammalian cells. Non-limiting examples include HEK cells, such as HEK-293 cells.

In certain embodiments of the presently disclosed subject matter, rAAV vector particles comprise a transgene that encodes a nucleic acid selected from the group consisting of a siRNA, an antisense molecule, miRNA a ribozyme and a shRNA.

In certain embodiments of the presently disclosed subject matter, rAAV vector particles comprise a transgene that encodes a gene product selected from the group consisting of insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietins, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor a (TGFa), platelet - derived growth factor (PDGF), insulin growth factors I and II (IGF- 1 and IGF - II), TGFB, activins, inhibins, bone morphogenic protein (BMP), nerve growth factor (NGF), brain - derived neurotrophic factor (BDNF), neurotrophins NT - 3 and NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line derived neurotrophic factor ( GDNF ), neurturin, agrin, netrin - 1 and netrin - 2, hepatocyte growth factor (HGF), ephrins, noggin, sonic hedgehog and tyrosine hydroxylase. In certain embodiments of the presently disclosed subject matter, rAAV vector particles comprise a transgene that encodes a gene product selected from the group consisting of thrombopoietin (TPO), interleukins (IL-1 through IL-17), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factors a and P, interferons a, P, and y, stem cell factor, flk-2/flt3 ligand, IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, class I and class II MHC molecules.

In certain embodiments of the presently disclosed subject matter, the rAAV vector particles comprise a transgene encoding a protein useful for correction of in born errors of metabolism selected from the group consisting of carbamoyl synthetase I , ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarylacetacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor V, factor VIII, factor IX, cystathione beta-synthase, branched chain ketoacid decarboxylase, albumin, isovaleryl-coA dehydrogenase, propionyl COA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase, insulin, betaglucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, RPE65, Flprotein, T-protein, a cystic fibrosis transmembrane regulator (CFTR) sequence, and a dystrophin cDNA sequence.

In certain embodiments of the presently disclosed subject matter, the rAAV vector particles comprise a transgene that encodes Factor VIII or Factor IX.

In certain embodiments of the presently disclosed subject matter, the AAV is AAV-GAA. In certain embodiments, AAV-GAA is a vector of the RHM4-1 capsid prepared (as shown in U.S. Patent No. 9,840,719) containing a polynucleotide sequence that, when transcribed, can be translated into GAA (prepared as described in US20210222141, which is incorporated herein by reference in its entirety).

In certain embodiments of the presently disclosed subject matter, the AAV is AAV-HemA. In certain embodiments, AAV-HemA is a vector of the LK03 capsid prepared (as shown in U.S. Patent No. 9,169,299) containing a polynucleotide sequence then, when transcribed, can be translated into BDD-FVIII (prepared as described in PCT/US2015/045142, which is incorporated herein by reference in its entirety). 4. Exemplary Methods

As disclosed herein, the instant disclosure provides compositions and methods for the purification of rAAV vector capsids. In certain embodiments, the methods of the present disclosure comprise: (a) contacting a sample comprising rAAV vector particles to an AAV affinity chromatography composition to produce a capture step eluate comprising rAAV vector particles; (b) contacting said capture step eluate to a first polishing chromatography composition to produce a first polishing eluate comprising rAAV vector particles; and (c) contacting said first polishing eluate to a second polishing chromatography composition to produce a second polishing eluate comprising differentially separated rAAV vector capsid variants. In certain of such embodiments, the capture step eluate and/or the first polishing eluate is incubated in a buffer solution comprising about 50 mM to about 100 mM Tris at a pH of about 4.5 to about 9.0, and a conductivity of about 2.5 mS/cm to about 15 mS/cm. In certain embodiments, the incubation time is between about 1 hour to about 12 hours.

In certain embodiments, the first polishing chromatography composition and the second polishing chromatography composition are selected from distinct separation strategies, e.g., strong AEX chromatography; strong CEX chromatography; HIC; MMC; and weak AEX chromatography. In certain embodiments, the first polishing chromatography composition and the second polishing chromatography composition are selected from the same separation strategy, (e.g., both compositions are strong AEX chromatography compositions) but comprise distinct ligands (e.g., distinct fixed ions in the case of IEX chromatography compositions).

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is selected from the group consisting of: a strong AEX chromatography composition (e.g., Poros XQ, Poros HS); a strong CEX chromatography composition; a MMC composition; a HIC composition, and a weak AEX chromatography composition; while the second polishing chromatography composition is selected from the group consisting of: a weak AEX chromatography composition; a strong AEX chromatography composition; a strong CEX chromatography composition; a HIC composition; and a MMC composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a strong AEX chromatography composition, and the second polishing chromatography composition is a weak AEX chromatography composition. In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a first strong AEX chromatography composition, and the second polishing chromatography composition is a second, distinct, strong AEX chromatography composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a strong AEX chromatography composition, and the second polishing chromatography composition is a HIC chromatography composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a strong AEX chromatography composition, and the second polishing chromatography composition is a MMC composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a strong CEX chromatography composition, and the second polishing chromatography composition is a weak AEX chromatography composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a strong CEX chromatography composition, and the second polishing chromatography composition is a strong AEX chromatography composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a strong CEX chromatography composition, and the second polishing chromatography composition is a HIC chromatography composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a strong CEX chromatography composition, and the second polishing chromatography composition is a MMC composition. In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a MMC composition, and the second polishing chromatography composition is a weak AEX chromatography composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a MMC composition, and the second polishing chromatography composition is a strong AEX chromatography composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a MMC composition, and the second polishing chromatography composition is a HIC chromatography composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a HIC chromatography composition, and the second polishing chromatography composition is a weak AEX chromatography composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a HIC chromatography composition, and the second polishing chromatography composition is a strong AEX chromatography composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the first polishing chromatography composition is a HIC chromatography composition, and the second polishing chromatography composition is a MMC composition.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and a capsid variant is differentially separated to greater than 90% purity.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and the capture step eluate and/or the first polishing eluate is diluted with a Tris buffer, wherein said Tris buffer comprises:(i) about 50 mM to about 150 mM Tris; (ii) a pH of about 4.5 to about 9.0; and (iii) a conductivity of about 6 mS/cm to 16 mS/cm. In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and contacting of the first and/or second polishing chromatography composition comprises equilibrating and/or washing said composition with a Tris buffer, wherein said Tris buffer comprises: (i) about 50 mM to about 150 mM Tris; (ii) a pH of about 7.5 to about 9.0; and (iii) a conductivity of about 6 mS/cm to 16 mS/cm.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and said rAAV vector capsid variants are eluted from said first and/or second polishing chromatography composition by contacting said composition with a salt-based elution buffer comprising: (i) about 50 mM to about 150 mM Tris; (ii) a pH of about 7.5 to about 9.0; and (iii) a conductivity of about 10 mS/cm to about 30 mS/cm.

In certain embodiments, the purification methods disclosed herein comprise at least two polishing chromatography steps to achieve the differential separation of rAAV vector capsid variants and said rAAV vector capsid variants are eluted from said first and/or second polishing chromatography composition by contacting said composition with a pH-based elution buffer comprising: (i) about 50 mM to 200 mM sodium acetate; (ii) a pH of about 3.0 to about 6.0; and (iii) a NaCl concentration of about 150 mM or greater.

5. Exemplary Buffers

5.1 Equilibration, Sample loadins, and Wash buffer (TRIS)

In certain embodiments of the presently disclosed subject matter, the chromatography compositions employed in the differential separation of rAAV capsid variants is equilibrated and/or washed with a Tris buffer. In certain embodiments of the presently disclosed subject matter, a sample or eluate comprising rAAV particles is diluted in a Tris buffer prior to contacting said rAAV particles with a strong AEX chromatography composition, a weak AEX chromatography composition, a strong CEX chromatography composition, an MMC chromatography composition, and/or an HIC chromatography composition.

In certain embodiments of the presently disclosed subject matter, the concentration of Tris in such a buffer is in a range of about 75 mM to about 125 mM, inclusive. In certain embodiments of the presently disclosed subject matter, the Tris buffer has a pH from about 7.5 to about 9.0, inclusive. In certain embodiments of the presently disclosed subject matter, the Tris buffer has a conductivity of about 6 mS/cm to 16 mScm, inclusive, which can, in certain embodiments, be adjusted with NaCl. In certain embodiments of the presently disclosed subject matter, the Tris buffer comprises at least one chaotropic salt. For example, but not by way of limitation, such chaotropic salt can be selected from the group consisting of ammonium sulfate and tetramethyl ammonium chloride, however, one of skill would appreciate the other chaotropic salts that can be used in connection with the subject matter disclosed herein. In certain embodiments of the presently disclosed subject matter, the Tris buffer can be free of MgC12, CaC12, and/or other metal ions. In certain embodiments of the presently disclosed subject matter, the Tris buffers comprise about 0.001% of Koliphor.

In certain embodiments of the presently disclosed subject matter, the equilibration, sample loading, and/or wash buffer is a Tris buffer, wherein the Tris buffer: i) comprises a concentration of about 50 mM to about 100 mM of Tris, e.g., about 100 mM; ii) is at a pH of about 8.5; iii) has a conductivity of about 6 mS/cm to 16 mS/cm (adjusted with NaCl); iv) comprises at least one chaotropic salt (e.g., ammonium sulfate, tetramethyl ammonium chloride); v) is free of MgC12, CaC12, and/or other metal ions; and vi) comprises 0.001% Koliphor.

5.2 Salt-based Elution Bu ffer

In certain embodiments of the presently disclosed subject matter, rAAV vector particles are eluted from a strong anion exchange column, a weak anion exchange column, a strong cation exchange column, a mixed mode column, and/or a hydrophobic interaction chromatography column with a salt-based elution buffer. In certain embodiments of the presently disclosed subject matter, a salt-based elution buffer comprises a concentration of a Tris in a range of about 75-125 mM, inclusive. In certain embodiments of the presently disclosed subject matter, a salt-based elution buffer has a pH from about 7.5 to about 9.0, inclusive. In certain embodiments of the presently disclosed subject matter, a Salt-based elution buffer has a conductivity of about 10 mS/cm to 30 mS/cm, inclusive, which is adjusted with NaCl or an equivalent salt. In certain embodiments of the presently disclosed subject matter, a salt-based elution buffer is essentially free of MgC12, CaC12, and other metal ions.

In certain embodiments of the presently disclosed subject matter, rAAV vector particles are eluted from a strong anion exchange column, a weak anion exchange column, a strong cation exchange column, a mixed mode column, and/or a hydrophobic interaction chromatography column with a Salt-based elution buffer, wherein the Salt-based elution buffer: i) comprises a concentration of about 50 mM to about 150 mM of Tris, preferable 100 mM; ii) is at a pH of about 8.5; iii) has a conductivity of about 10 mS/cm to 30 mS/cm and is adjusted with NaCl or equivalent salt; and iv) is free of MgC12, CaC12, and other metal ions. 5.3 pH-based elution buffer

In certain embodiments of the presently disclosed subject matter, rAAV vector particles are eluted from a strong anion exchange column, a weak anion exchange column, a strong cation exchange column, a mixed mode column, and/or a hydrophobic interaction chromatography column with a pH-based elution buffer. In certain embodiments of the presently disclosed subject matter, a pH-based elution buffer comprises a concentration of a sodium acetate or equivalent in a range of about 15 mM to about 125 mM, inclusive. In certain embodiments of the presently disclosed subject matter, a pH-based elution buffer has a pH from about 6.5 to about 2.5, inclusive. In certain embodiments of the presently disclosed subject matter, a pH-based elution buffer comprises a NaCl concentration of about 250 mM or higher.

In certain embodiments of the presently disclosed subject matter, rAAV vector particles are eluted from a strong anion exchange chromatography composition, a strong cation exchange chromatography composition, and/or a mixed mode chromatography composition, with a pH- based elution buffer, wherein the pH-based elution buffer: i) comprises a concentration of about 100 mM of sodium acetate; ii) is at a pH of about 3.0 to 6.0, inclusive; and iii) comprises a concentration of NaCl about 250 mM or greater.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the presently disclosed subject matter, suitable methods and materials are described herein.

All applications, publications, patents and other references, GenBank citations and ATCC citations cited herein are incorporated by reference in their entirety. In case of conflict, the specification, including definitions, will control.

All of the features disclosed herein may be combined in any combination. Each feature disclosed in the specification may be replaced by an alternative feature serving a same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, disclosed features (e.g., nucleic acid sequences, vectors, rAAV vectors, etc.) are an example of a genus of equivalent or similar features.

As used herein, the singular forms "a", "and," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "an AAV vector," or "AAV particle," includes a plurality of such AAV vectors and AAV particles, and reference to "a cell" or "host cell" includes a plurality of cells and host cells.

The term "about" as used herein means values that are within 10% (plus or minus) of a reference value. As used herein, all numerical values or numerical ranges include integers within such ranges and fractions of the values or the integers within ranges unless the context clearly indicates otherwise. Thus, to illustrate, reference to 80% or more identity, includes 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% etc., as well as 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, etc., 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, etc., and so forth.

Reference to an integer with more (greater) or less than includes any number greater or less than the reference number, respectively. Thus, for example, a reference to less than 100, includes 99, 98, 97, etc. all the way down to the number one (1); and less than 10, includes 9, 8, 7, etc. all the way down to the number one (1).

As used herein, all numerical values or ranges are inclusive. Further, all numerical values or ranges include fractions of the values and integers within such ranges and fractions of the integers within such ranges unless the context clearly indicates otherwise. Thus, to illustrate, reference to a numerical range, such as 1-10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2,

1.3, 1.4, 1.5, etc., and so forth. Reference to a range of 1-50 therefore includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., up to and including 50, as well as 1.1, 1.2, 1.3,

1.4, 1.5, etc., 2.1, 2.2, 2.3, 2.4, 2.5, etc., and so forth.

Reference to a series of ranges includes ranges which combine the values of the boundaries of different ranges within the series. Thus, to illustrate reference to a series of ranges, for example, of 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1,000, 1,000-1, 500, 1,500-2,000, 2,000-2,500, 2,500-3,000, 3,000-3,500, 3,500-4,000, 4,000-4,500, 4,500-5,000, 5,500-6,000, 6,000-7,000, 7,000-8,000, or 8,000-9,000, includes ranges of 10-50, 50-100, 100-1,000, 1,000-3,000, 2,000-4,000, etc.

The invention is generally disclosed herein using affirmative language to describe the numerous embodiments and aspects. The invention also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, or procedures. For example, in certain embodiments or aspects of the invention, materials and/or method steps are excluded. Thus, even though the invention is generally not expressed herein in terms of what the invention does not include aspects that are not expressly excluded in the invention are nevertheless disclosed herein.

A number of embodiments of the invention have been described. Nevertheless, one skilled in the art, without departing from the spirit and scope of the invention, can make various changes and modifications of the invention to adapt it to various usages and conditions. Accordingly, the following examples are intended to illustrate but not limit the scope of the invention claimed. EXAMPLES

Example 1

Exemplary chromatography polishing step 1 : Strong anion exchange Poros XQ; Exemplary chromatography polishing step 2: Weak anion exchange BIA monolith DEAE.

Step 1 : Strong anion exchange on Poros XQ. Separation of empty and non-empty LK03 capsid variants on Poros XQ (Figure 2A). Equilibration, sample loading, and wash buffer: 100 mM Tris pH 8.5, 50 mM NaCl, 15 mM ammonium sulfate (conductivity 10.5 mS/cm). Elution gradient buffer: 100 mM Tris pH 8.5, 250 mM NaCl. Sample loading buffer: 100 mM Tris pH 8.5, 50 mM NaCl, 15 mM tetramethyl ammonium chloride (conductivity 10.5 mS/cm). The flowthrough (Peak 1) is empty capsids, while the elution contains several non-empty capsids (Peak 2, Peak 3, Peak 4, and Peak 5) and a second population of empty capsid.

Step 2: Weak anion exchange - Monolith BIA DEAE. Separation of empty and non-empty capsid variants on Monolith BIA DEAE from the samples eluted from Poros XQ in Stepl . Peak 2 (P2), Peak 3 (P3), and Peak 4 (P4) from Figure 2B were collected and further resolved into 14 peaks on Monolith BIA DEAE (P2a-P2f, P3a-P3e, P4a-P4c). Equilibration and wash buffer: 100 mM Tris pH 8.5, 80 mM NaCl (conductivity 9.5 mS/cm). Elution gradient buffer: 100 mM Tris pH 8.5, 250 mM NaCl. Sample loading buffer: 100 mM Tris pH 8.5, 40 mM NaCl, 15 mM tetramethyl ammonium chloride (conductivity 9.5 mS/cm). The flowthrough is empty capsids while the elution contains several non-empty capsids (P2a-P2f, P3a-P3e, P4a-P4c).

AEX for AAV separation is a well-established mode of chromatography. Resin capacity and resolution between empty and full capsid (translates to % empty capsid removal) is a preferable first step to remove the maximum amount of empty capsid in the flowthrough. Monolith BIA DEAE resolves the maximum number of variants as distinct peaks in the second step, compared to other examples with the best purity. The potency of the variants separated in Figure 2A and 2B relative to the lowest peak potency was measured using an in vitro cell-based assay. The cell-based assay measures transgene expression. Methods to assay transgene expression are well known and are specific to the particular transgene utilized. The relative potency, which is ratio of the potency of the test samples to that of a reference (e.g., the lowest peak potency) is presented in the form of a percentage. The variants shown in Figure 2A and 2B were also characterized with mass photometry and the percentage breakdown of the capsid containing no transgene (empty), incomplete transgene (partial) and the full-length transgene (full) are shown in Figure 2C. Example 2

Exemplary chromatography polishing step 1 : Strong anion exchange Poros XQ; Exemplary chromatography polishing step 2: Strong anion exchange BIA monolith QA. Step 1 : Same as Example 1.

Step 2: Separation of empty and non-empty capsid variants on Monolith BIA QA from the sample eluted from Poros XQ Peak2 (Figure 3). Equilibration and wash buffer: 100 mM Tris pH 8.5, 80 NaCl mM (conductivity 9.5 mS/cm). Elution gradient buffer: 100 mM Tris pH 8.5, 250 mM NaCl. Sample loading buffer: 100 mM Tris pH 8.5, 40 mM NaCl, 15 mM tetramethyl ammonium chloride (conductivity 9.5 mS/cm). The flowthrough is empty capsids (6), while the elution contains several non-empty capsids (7-9).

AEX for AAV separation is a well-established mode of chromatography. Although Monolith BIA QA does not resolve as many capsid variants as Monolith BIA DEAE, it resolves different capsid variants in 4 peak pools.

Example 3

Exemplary chromatography polishing step 1 : Strong cation exchange Poros XS; Exemplary chromatography polishing step 2: Strong anion exchange Poros XQ.

Step 1 : Separation of empty and non-empty LK03 capsid variants on Poros XS (Figure 4A). Equilibration, sample loading, and wash buffer: 100 mM Tris pH 8.5, 140 mM NaCl (conductivity 15.5 mS/cm). Elution gradient buffer: 100 mM Tris pH 8.5, 250 mM NaCl. The flowthrough is empty capsids (1), while the elution peak contains several non-empty and empty capsids (2).

Step 2: Separation of empty and non-empty capsid variants on Poros XQ from the samples eluted from Poros XS peak 2 (Figure 4B). Equilibration, sample loading 100 mM Tris pH 8.5, 45 mM NaCl, 15 mM ammonium sulfate (conductivity 10 mS/cm). Elution gradient buffer: 100 mM Tris pH 8.5, 250 mM NaCl. Sample loading buffer: 100 mM Tris pH 8.5, 50 mM NaCl, 15 mM tetramethyl ammonium chloride (conductivity 10.5 mS/cm). The flowthrough and step elution contains the empty capsid (3), while the elution contains several non-empty as well as the second population of empty capsid (4-6).

CEX for AAV separation allows for loading at higher conductivity, compared to AEX (15.5 mS/cm vs. 9.5 mS/cm), which promotes better capsid stability and, subsequently, a higher recovery. This also helps resolve peaks 4, 5, and 6 (Figure 4B) with better purity. However, the empty capsid variant resolution is better with strong anion exchange (Poros XQ) as the first chromatography polishing step.

Example 4

Exemplary chromatography polishing step 1 : Strong anion exchange Poros XQ; Exemplary chromatography polishing step 2: Strong anion exchange BIA monolith QA HR.

Step 1 : Same as example 1. Step 2: Separation of Poros XQ Peak2 eluate on Monolith BIA QA HR (Figure 5). Equilibration and wash buffer: 100 mM Tris pH 8.5, 80 mM NaCl (conductivity 9.5 mS/cm). Elution gradient buffer: 100 mM Tris pH 8.5, 250 mMNaCl. Sample loading buffer: 100 mM Tris pH 8.5, 40 mM NaCl, 15 mM tetramethyl ammonium chloride (conductivity 9.5 mS/cm). The flowthrough is empty capsids (6), while the elution contains several non-empty and empty capsids (7-10).

BIA monolith QA HR is a variation of the monolith BIA QA column that yields 5 different peak pools of different capsid variants.

Example 5

Exemplary chromatography polishing step 1 : Strong anion exchange Poros XQ; Exemplary chromatography polishing step 2: Strong anion exchange membrane Q (e.g., Sartobind Q, Mustang Q)

Step 1 : Same as example 1.

Step 2a: Separation of Poros XQ Peak2 eluate on Membrane Sartobind Q (Figure 6A). Equilibration and wash buffer: 100 mM Tris pH 8.5, 80 mM NaCl (conductivity 9.5 mS/cm). Elution gradient buffer: 100 mM Tris pH 8.5 250 mM NaCl. Sample loading buffer: 100 mM Tris pH 8.5 40 mM NaCl 15 mM tetramethyl ammonium chloride (conductivity 9.5 mS/cm). The flowthrough is the empty capsid (6), while the elution contains several non-empty capsids and empty capsid (7-9).

Step 2b: Separation of Poros XQ Peak2 eluate on Membrane Mustang Q (Figure 6B). Equilibration and wash buffer: 100 mM Tris pH 8.5, 80 mM NaCl (conductivity 9.5 mS/cm). Elution gradient buffer: 100 mM Tris pH 8.5, 250 mMNaCl. Sample loading buffer: 100 mM Tris pH 8.5, 40 mM NaCl, 15 mM tetramethyl ammonium chloride (conductivity 9.5 mS/cm). The flowthrough is the empty capsid (6), while the elution contains several non-empty capsids (7 -8).

Membrane chromatography allows for higher throughput operation and lower AAV-resin contact time, which is favorable for capsid stability.

Example 6

Exemplary chromatography polishing step 1 : Mixed mode (anion and hydrophobic) resin (Capto Adhere ImpRes); Exemplary chromatography polishing step 2: Weak anion exchange BIA monolith DEAE.

Step 1 : Separation of affinity chromatography eluate on mixed mode Capto Adhere ImpRes (Figure 7A). Equilibration and wash buffer: 100 mM Tris pH 8.5, 150 mM NaCl (conductivity 15 mS/cm). Elution step buffers: (1) 100 mM acetate pH 5.5, 250 mM NaCl (2) 20 mM acetate pH 3, 500 mM NaCl. Sample loading buffer: 100 mM Tris pH 8.5, 135 mM NaCl, 15 mM tetramethyl ammonium chloride (conductivity 15 mS/cm). The flowthrough is the empty capsid (1), while the elution contains several non-empty capsids and the empty capsid (2-3).

Step 2: Separation of the Capto adhere Peak2 elution sample on Monolith BIA DEAE (Figure 7B). Equilibration and wash buffer: 100 mM Tris pH 8.5, 80 mM NaCl (conductivity 9.5 mS/cm). Elution gradient buffer: 100 mM Tris pH 8.5, 250 mM NaCl. Sample loading buffer: 100 mM Tris pH 8.5, 40 mMNaCl, 15 mM tetramethyl ammonium chloride (conductivity 9.5 mS/cm). The flowthrough is empty capsid (4), while the elution contains three non-empty capsids as three distinct peaks (5-7).

MMC for AAV separation allows for loading at higher conductivity, compared to AEX (15 mS/cm vs. 9.5 mS/cm), which promotes better capsid stability for peak 1 and 2 and, subsequently, a higher recovery but at lower pH of Peak 3 may lead to poor stability of this particular species (Figure 7A).

Example 7

Exemplary chromatography polishing step 1 : Strong anion exchange Poros XQ; Exemplary chromatography polishing step 2: Hydrophobic Interaction Chromatography, HIC (e.g., Capto butyl ImpRes, Capto Phenyl ImpRes).

Step 1 : Same as example 1.

Step 2: Capto Butyl ImpRes (Figure 8A) or Capto Phenyl ImpRes (Figure 8B). Separation of Poros XQ Peak 2 on HIC resin. Equilibration and wash buffer: 100 mM Tris pH 8.5, 80 mM NaCl (conductivity 9.5 mS/cm) 0.001% Koliphor. Elution gradient buffer: 100 mM Tris pH 8.5, 250 mM NaCl, 0.001% Koliphor. Sample loading buffer: 100 mM Tris pH 8.5, 80 mM NaCl, 0.001% Koliphor. The flowthrough (6) and elution (7) contain different full and empty capsids variants.

HIC selects one variant effectively. This is different from the traditional mode of HIC operation, where binding is carried out at higher ionic strength while elution is carried out at lower ionic strength. In this example, the mode is reversed, binding at a lower salt while elution is at a higher salt.

Example 8

Exemplary chromatography polishing step 1 : Strong anion exchange Poros XQ; Exemplary chromatography polishing step 2: mixed mode (Prima S).

Step 1 : Same as example 1.

Step 2: Separation of Poros XQ Peak 2 on Monolith BIA PrimaS (Figure 9). Equilibration and wash buffer: 100 mM Tris pH 8.5, 80 mM NaCl (conductivity 9.5 mS/cm). Elution gradient buffer: 100 mM Tris pH 8.5, 250 mM NaCl. Sample loading buffer: 100 mM Tris pH 8.5, 40 mM NaCl, 15 mM tetramethyl ammonium chloride (conductivity 9.5 mS/cm). The flowthrough is empty capsids (6), while the elution contains non-empty capsids as four peaks (7-10).

Prima S provides an alternative mixed mode resin in step 2 to resolve capsid variants in 5 different peak pools (6-10) (Figure 9), although recovery is poor compared to example 1 step 2. Example 9

Analytical scale capsid heterogeneity analysis. Figures 10A-10C demonstrates the capsid heterogeneity recovered by AAV affinity capture alone (Figure 10A) versus a Poros XQ (Figure 10B) and two-step approach (Figure 10C).

Example 10 Exemplary separation for an AAV of RHM4-1 (capsid) with strong cation exchange Poros

XS. Figures 11 A-l IB demonstrate the separation with three resolved peaks 1, 2 &3 on a Poros XS resin using both pH and salt gradient. Equilibration and wash buffer: 25 mM Acetate pH 6.2 Elution gradient buffer: 100 mM Acetate pH 5.5, 300 mM NaCl (Figure 11 A). Salt gradient - Equilibration and wash buffer: 25 mM Acetate pH 5.5 and elution gradient buffer: lOOmM Acetate pH 5.5, 300 mM NaCl (Figure 11B).

Claims

WHAT IS CLAIMED IS:

1. A method for differentially separating recombinant adeno-associated (rAAV) vector capsid variants, said method comprising:

(a) contacting a sample comprising rAAV vector particles to an AAV affinity chromatography composition to produce a capture step eluate comprising rAAV vector particles;

(b) contacting said capture step eluate to a first polishing chromatography composition to produce a first polishing eluate comprising rAAV vector particles; and

(c) contacting said first polishing eluate to a second polishing chromatography composition to produce a second polishing eluate comprising differentially separated rAAV vector capsid variants.

2. A method according to claim 1, wherein the capture step eluate and/or the first polishing eluate is incubated in a buffer solution comprising about 50 mM to about 150 mM Tris at a pH of about 4.5 to about 9.0 and a conductivity of about 2.5 mS/cm to about 15 mS/cm.

3. A method according to claim 2, wherein the incubation time is between about 1 hour to about 12 hours.

4. A method according to claim 1, wherein the first polishing chromatography composition and the second polishing chromatography composition are different.

5. A method according to claim 1, wherein the first polishing chromatography composition is selected from the group consisting of: a strong anion exchange (AEX) chromatography composition; a mixed mode chromatography (MMC) composition; a strong cation exchange (CEX) chromatography composition; hydrophobic interaction chromatography (HIC) composition; and a weak AEX chromatography composition.

6. A method according to claim 1, wherein the second polishing chromatography composition is selected from the group consisting of: a weak AEX chromatography composition; a strong AEX chromatography composition; a strong CEX chromatography composition; a hydrophobic interaction chromatography composition; and a MMC composition.

7. A method according to claim 1, wherein: (i) the first polishing chromatography composition is selected from the group consisting of a strong AEX chromatography composition; a strong CEX chromatography composition; a MMC composition; a HIC composition; and a weak AEX chromatography composition; and

(ii) the second polishing chromatography composition is selected from the group consisting of a weak AEX chromatography composition; a strong AEX chromatography composition; a strong CEX chromatography composition; a HIC composition; and a MMC composition.

8. A method according to claim 1, wherein first polishing chromatography composition is a strong AEX chromatography composition, and the second polishing chromatography composition is a weak AEX chromatography composition.

9. A method according to claim 1, wherein first polishing chromatography composition is a first strong AEX chromatography composition, and the second polishing chromatography composition is a second, distinct, strong AEX chromatography composition.

10. A method according to claim 1, wherein first polishing chromatography composition is a strong AEX chromatography composition, and the second polishing chromatography composition is a HIC chromatography composition.

11. A method according to claim 1, wherein first polishing chromatography composition is a strong AEX chromatography composition, and the second polishing chromatography composition is a MMC composition.

12. A method according to claim 1, wherein first polishing chromatography composition is a strong CEX chromatography composition, and the second polishing chromatography composition is a weak AEX chromatography composition.

13. A method according to claim 1, wherein first polishing chromatography composition is a strong CEX chromatography composition, and the second polishing chromatography composition is a strong AEX chromatography composition.

14. A method according to claim 1, wherein first polishing chromatography composition is a strong CEX chromatography composition, and the second polishing chromatography composition is a HIC chromatography composition.

15. A method according to claim 1, wherein first polishing chromatography composition is a strong CEX chromatography composition, and the second polishing chromatography composition is a MMC composition.

16. A method according to claim 1, wherein first polishing chromatography composition is a MMC composition, and the second polishing chromatography composition is a weak AEX chromatography composition.

17. A method according to claim 1, wherein first polishing chromatography composition is a MMC composition, and the second polishing chromatography composition is a strong AEX chromatography composition.

18. A method according to claim 1, wherein first polishing chromatography composition is a MMC composition, and the second polishing chromatography composition is a HIC chromatography composition.

19. A method according to claim 1, wherein first polishing chromatography composition is a HIC chromatography composition, and the second polishing chromatography composition is a weak AEX chromatography composition.

20. A method according to claim 1, wherein first polishing chromatography composition is a HIC chromatography composition, and the second polishing chromatography composition is a strong AEX chromatography composition.

21. A method according to claim 1, wherein first polishing chromatography composition is a HIC chromatography composition, and the second polishing chromatography composition is a MMC composition.

22. A method according to claim 1, wherein a capsid variant is differentially separated to greater than 90% purity.

23. A method according to claim 1, wherein the capture step eluate and/or the first polishing eluate is diluted with a Tris buffer, wherein said Tris buffer comprises:

(i) about 50 mM to about 150 mM Tris;

(ii) a pH of about 4.5 to about 9.0; and (iii) a conductivity of about 6 mS/cm to 16 mS/cm.

24. A method according to claim 1, wherein contacting of the first and/or second polishing chromatography composition comprises equilibrating and/or washing said composition with a Tris buffer, wherein said Tris buffer comprises:

(i) about 50 mM to about 150 mM Tris;

(ii) a pH of about 4.5 to about 9.0; and

(iii) a conductivity of about 6 mS/cm to 16 mS/cm.

25. A method according to claim 1, wherein said rAAV vector particles are eluted from said first and/or second polishing chromatography composition by contacting said composition with a salt-based elution buffer comprising:

(i) about 50 mM to about 150 mM Tris;

(ii) a pH of about 4.5 to about 9.0; and

(iii) a conductivity of about 10 mS/cm to about 30 mS/cm.

26. A method according to claim 1, wherein said rAAV vector particles are eluted from said first and/or second polishing chromatography composition, wherein said first and/or second polishing chromatography composition is selected from a strong AEX chromatography composition, a strong CEX chromatography composition, or a MMC composition, by contacting said first and/or second polishing chromatography composition with a pH-based elution buffer comprising:

(i) about 50 mM to 200 mM sodium acetate;

(ii) a pH of about 3.0 to about 6.0; and

(iii) a NaCl concentration of about 250 mM or greater.

27. A method according to claim 1, wherein the differentially separated rAAV vector capsid variants of step (c) have a different relative potency than the rAAV vector particles contacted in step (a).

28. A method according to claim 1, wherein the differentially separated rAAV vector capsid variants of step (c) have a higher relative potency than the rAAV vector particles contacted in step (a).

29. A method according to claim 1, wherein the differentially separated rAAV vector capsid variants of step (c) have a different relative potency than the rAAV vector particles produced in step (b).

30. A method according to claim 1, wherein the differentially separated rAAV vector capsid variants of step (c) have a higher relative potency than the rAAV vector particles produced in step (b).