WO2021158915A1

WO2021158915A1 - Recombinant adeno-associated virus compositions and methods for producing and using the same

Info

Publication number: WO2021158915A1
Application number: PCT/US2021/016819
Authority: WO
Inventors: Stephen M. SOLTYS
Original assignee: Precision Biosciences, Inc.
Priority date: 2020-02-06
Filing date: 2021-02-05
Publication date: 2021-08-12

Abstract

The present invention encompasses improved methods for separating full recombinant adeno-associated virus (rAAV) particles from empty rAAV particles using anion exchange chromatography with an isocratic elution gradient of magnesium chloride. The improved methods allow for the production of viral compositions comprising optimized percentages of full rAAV particles, wherein the AAV can be of any serotype. The optimized viral compositions find use in delivering genes of interest to cells by contacting the cells with the viral compositions.

Description

RECOMBINANT ADENO-ASSOCIATED VIRUS COMPOSITIONS AND METHODS FOR PRODUCING AND USING THE SAME

FIELD OF THE INVENTION

The invention relates to the field of virology, molecular biology and recombinant nucleic acid technology. In particular, the invention relates to compositions comprising recombinant adeno-associated virus (rAAV) with optimized levels of full, genome-containing vims and methods for producing such optimized viral compositions. The optimized viral compositions are useful in methods for gene delivery.

BACKGROUND OF THE INVENTION

Viral vectors are used to deliver genetic material into host cells for various applications, including gene editing and gene therapy. Adeno-associated vims (AAV) is a small, replication-defective vims belonging to the family of Parvoviridae that has shown promise for use in delivering genes to both dividing and quiescent cells. AAV is not pathogenic to humans, only replicates in the presence of a helper vims, and transgene expression by rAAV is potentially long lasting. Although the packaging capacity is limited to approximately 4.5 kb due to its small size, its many advantages make it a preferred tool for gene delivery purposes.

Methods for purifying adeno-associated vims particles have been reported, including affinity chromatography using antibodies specific for AAV capsid proteins. Affinity chromatography, although highly selective for the AAV capsid, cannot discriminate between a full (genome-containing) viral particle and an empty viral particle. Increased levels of empty viral particles within a rAAV preparation reduces therapeutic efficacy and could enhance host immune responses. Thus, various methods for separating empty and full rAAV particles in order to enrich viral compositions for full rAAV viral particles have been proposed. Empty rAAV viral particles can be effectively separated from full rAAV particles by density gradient centrifugation using cesium chloride or iodixanol gradients based on the differing densities between the full (1.40 g/cm³) and empty (1.32 g/cm³) viral particles. However, these methods are not easily scalable and are costly.

Other proposed methods for separating empty and full rAAV particles take advantage of the subtle difference in surface charge between a full rAAV particle (average pi of about 5.9) and empty rAAV particle (average pi of about 6.3) by using ion exchange chromatography (IEX). Although the separation of rAAV empty and full particles using column chromatography with either monoliths or packed resin beds of various media for cation or anion exchange has been reported, generally, the process is only successful for a single AAV serotype and cannot be easily adapted to other serotypes.

Thus, there remains a need for scalable methods for purifying full rAAV particles that can be used for the purification of full viral particles of multiple AAV serotypes.

SUMMARY OF THE INVENTION

The present disclosure provides methods for effectively separating empty and full recombinant adeno-associated virus (rAAV) particles from viral samples. The methods rely on the use of an isocratic elution gradient of magnesium chloride as the sole salt to elute and separate the empty and full rAAV particles within a viral sample from an anion exchange chromatography medium. The improved methods allow for the separation of empty and full viral particles of any AAV serotype, including AAV5, AAV6 and AAV8 and the production of viral compositions enriched for full rAAV particles. Thus, in another aspect, the present disclosure provides viral compositions comprising recombinant adeno-associated virus (rAAV) with enriched levels of full, genome-containing virus. In some embodiments, the viral composition comprises a higher ratio of full rAAV particles to empty rAAV particles when compared to a viral composition produced using another method of full rAAV purification.

The optimized viral compositions are useful in methods for gene delivery and gene therapy. Thus, in another aspect, the present disclosure provides pharmaceutical compositions comprising the enriched viral compositions and a pharmaceutically acceptable carrier. In yet another aspect, methods for delivering a gene of interest by contacting a cell with the enriched viral compositions or pharmaceutical compositions comprising the same are provided, wherein the viral compositions comprise full rAAV particles comprising the gene of interest. In still another aspect, the present disclosure provides a method for treating a disease, wherein said method comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a viral composition produced by the presently disclosed methods, wherein expression of the gene of interest treats the disease.

In yet another aspect, the present disclosure provides a viral composition produced by the presently disclosed methods comprising full rAAV particles for use as a medicament. The present disclosure further provides the use of a viral composition produced by the presently disclosed methods comprising full rAAV particles in the manufacture of a medicament for delivering a gene of interest for treating a disease in a subject in need thereof.

In some embodiments, the full rAAV particles comprise a gene of interest that can encode an engineered nuclease, such as an engineered meganuclease, a TALEN, a compact TALEN, a zinc finger nuclease, a megaTAL, or a CRISPR system nuclease. In some embodiments, the full rAAV particles comprise a gene of interest that can encode a chimeric antigen receptor or exogenous T cell receptor.

In certain embodiments, the anion exchange chromatography medium used in the presently disclosed methods comprises a monolithic column. The monolithic column can comprise methacrylate, such as poly(glycidyl methacrylate-co-ethylene dimethacrylate). In particular embodiments, the anion exchange chromatography medium can comprise a quaternary amine functional group, such as the quaternary amine of CIMmultus™ QA or CIM^® QA. In other embodiments, the anion exchange chromatography medium used in the presently disclosed methods comprises CIMmultus™ QA or CIM^® QA.

The viral sample can be loaded on the anion exchange chromatography medium in the presence of a first isocratic buffer comprising Bis-Tris propane. In some embodiments, the isocratic elution gradient comprises increasing the percentage of a second isocratic buffer to the first isocratic buffer, wherein the second isocratic buffer comprises Bis-Tris propane and magnesium chloride, and wherein the second isocratic buffer does not comprise one or more additional salts having a total concentration which affects separation of the empty rAAV particles and the full rAAV particles. In certain embodiments, the concentration of magnesium chloride in the second isocratic buffer is between 1 mM and 100 mM or between 20 mM and 60 mM. In some of these embodiments, the concentration of magnesium chloride in the second isocratic buffer is about 40 mM.

In certain embodiments, the concentration of Bis-Tris propane in the first isocratic and the second isocratic buffer is between 5 mM and 30 mM. In particular embodiments, the Bis-Tris propane is present in the first isocratic buffer and the second isocratic buffer at a concentration of about 20 mM. In some of those embodiments wherein the first isocratic buffer and the second isocratic buffer comprises Bis-Tris propane, the pH of the buffer is between 8 and 9. In particular embodiments, the pH of the first isocratic buffer and the second isocratic buffer is about 8.5.

In some embodiments, the first isocratic buffer used for the loading step (and wash step) comprises Bis-Tris propane at a concentration of about 20 mM and at a pH of about 8.5. In certain embodiments, the second isocratic buffer used for the isocratic elution gradient comprises Bis-Tris propane at a concentration of about 20 mM and at a pH of about 8.5 and magnesium chloride at a concentration of about 40 mM.

In those embodiments wherein the method comprises the separation of empty and full AAV6 particles, the elution of empty rAAV particles occurs at a conductivity of about 6.2 mS/cm and the elution of full rAAV particles occurs at a conductivity of about 7.8 mS/cm.

In some of these embodiments, the elution of empty rAAV particles occurs at about 58.5% second isocratic buffer and elution of full rAAV particles occurs at about 80% second isocratic buffer.

Methods for separating empty and full AAV5 particles can comprise the elution of empty rAAV particles at a conductivity of about 5.5 mS/cm and the elution of full rAAV particles at a conductivity of about 6.4 mS/cm. In some of these embodiments, the elution of empty rAAV particles occurs at about 50.0% second isocratic buffer and elution of full rAAV particles occurs at about 61% second isocratic buffer.

In those embodiments wherein the separation of empty and full AAV8 particles is desired, the elution of empty rAAV particles can occur at a conductivity of about 4.9 mS/cm and the elution of full rAAV particles can occur at a conductivity of about 7.0 mS/cm. In some of these embodiments, the elution of empty rAAV particles occurs at about 41.7% second isocratic buffer and the elution of full rAAV particles occurs at about 70% second isocratic buffer.

In certain embodiments, the methods further comprise loading the viral sample onto the anion exchange chromatography medium and washing the medium to remove the unbound fraction of the viral sample prior to elution. In some of these embodiments, prior to loading the viral sample onto the anion exchange chromatography medium, the pH of the viral sample is increased to between 8 and 9 with Bis-Tris propane. In particular embodiments, the pH of the viral sample is increased to about 8.5.

The conductivity points used to elute empty and full rAAV particles can be identified by performing a previous anion exchange chromatography wherein a fraction of the viral sample is loaded onto the anion exchange chromatography medium and eluted with a linear elution gradient of magnesium chloride, wherein the first step of the isocratic elution gradient for elution of empty rAAV particles comprises the conductivity at which absorbance at 280 nm is greater than absorbance at 260 nm and absorbance at 280 nm is at its peak ± up to 0.5 mS/cm, and wherein the second step of the isocratic elution gradient for the elution of full rAAV particles comprises the conductivity at which absorbance at 260 nm is greater than absorbance at 280 nm and absorbance at 260 nm is at its peak ± up to 0.5 mS/cm. The pH of the fraction of the viral sample that is loaded in this previous anion exchange chromatography step can also be increased to between 8 and 9 with Bis-Tris propane prior to loading, and in some embodiments to a pH of about 8.5. In such embodiments, the linear elution gradient does not comprise one or more additional salts having a total concentration which affects separation of the empty rAAV particles and the full rAAV particles.

In some embodiments, the linear elution gradient comprises increasing the percentage of a second linear buffer to a first linear buffer, wherein the second linear buffer comprises Bis-Tris propane and magnesium chloride, and wherein the second linear buffer does not comprise one or more additional salts having a total concentration which affects separation of the empty rAAV particles and the full rAAV particles. In certain embodiments, the concentration of magnesium chloride in the second linear buffer is between 1 mM and 100 mM or between 20 mM and 60 mM. In some of these embodiments, the concentration of magnesium chloride in the second linear buffer is about 40 mM.

In certain embodiments, the concentration of Bis-Tris propane in the first linear and the second linear buffer is between 5 mM and 30 mM. In particular embodiments, the Bis- Tris propane is present in the first linear buffer and the second linear buffer at a concentration of about 20 mM. In some of those embodiments wherein the first linear buffer and the second linear buffer comprises Bis-Tris propane, the pH of the buffer is between 8 and 9. In particular embodiments, the pH of the first linear buffer and the second linear buffer is about 8.5.

In particular embodiments, the viral sample that is used in either of the anion exchange chromatography steps comprises a host cell lysate. In other embodiments, the viral sample used in anion exchange chromatography is an enriched eluate from a previous affinity chromatography step of a host cell lysate, wherein the enriched eluate has been enriched for rAAV particles. Thus, in some of these embodiments, the method further comprises performing affinity chromatography of a host cell lysate using an affinity chromatography medium that is specific for AAV, eluting with a low pH buffer, and collecting the enriched eluate that is subsequently loaded onto the anion exchange chromatography medium.

The methods can further comprise collecting the enriched eluate from the anion exchange chromatography that comprises full rAAV particles based on the 260nm/280nm absorbance readings.

In certain embodiments, the presence of full rAAV particles within the eluate from affinity chromatography, the eluate from anion exchange chromatography, or both is confirmed by performing quantitative PCR with primers specific for viral genomic DNA or the gene of interest.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a chromatogram of an attempt at separating empty and full adeno- associated virus serotype 6 (AAV6) using the CIMmultus™ QA monolith followed by a linear sodium chloride elution gradient. The chromatogram provides the absorption of the eluate and flow-through at 260 nm (purple line) and 280 nm (blue line) in milli Absorption Units (mAU). Also provided is the conductivity shown in orange and the concentration of Buffer B shown in green.

Figure 2 provides a chromatogram of an attempt at separating empty and full AAV6 using the CIMmultus™ QA monolith followed by a linear low pH elution gradient. The chromatogram provides the absorption of the eluate and flow-through at 280 nm (blue line) in milli Absorption Units (mAU). Also provided is the conductivity shown in orange, the concentration of Buffer B shown in green, and the pH shown in purple.

Figure 3 shows a chromatogram demonstrating the separation of empty and full AAV6 using the CIMmultus™ QA monolith followed by a linear magnesium chloride elution gradient. The chromatogram provides the absorption of the eluate and flow-through at 260 nm (purple line) and 280 nm (blue line) in milli Absorption Units (mAU). Also provided is the conductivity shown in orange and the concentration of Buffer B shown in green. The separation crossover event of 260/280 absorption is shown in the circled area of the chromatogram.

Figure 4 provides a chromatogram of a broader linear magnesium chloride gradient used to elute empty and full AAV6 from the CIMmultus™ QA monolith in order to specifically isolate the conductivity point where UV260/280 crossover occurs. The chromatogram provides the conductivity in mS/cm as an orange line. Absorption of the eluate and flow-through at 260 nm and 280 nm is provided in milli Absorption Units (mAU) as the purple and blue line, respectively. Also provided is the concentration of Buffer B shown in green. The arrows indicate the conductivity range that was targeted in subsequent experiments.

Figure 5 provides a chromatogram of a narrower, more focused linear gradient of magnesium chloride to elute empty and full AAV6 from the CIMmultus™ QA monolith in order to specifically isolate the conductivity points that were targeted for an isocratic elution in subsequent experiments. Figures 6A-6C provide chromatograms of the separation of empty and full AAV6 particles from three samples using an isocratic gradient of magnesium chloride. A) Sample number 1. B) Sample number 2. C) Sample number 3.

Figure 7 provides a chromatogram of the separation of empty and full AAV5 particles using an isocratic gradient of magnesium chloride.

Figure 8 provides a chromatogram of the separation of empty and full AAV8 particles using an isocratic gradient of magnesium chloride.

Figure 9 provides an image of a dot blot demonstrating the presence of viral capsid proteins in fractions correlating to the peaks seen in the chromatograms of Figures 6-8 of AAV6, AAV5, and AAV8, respectively.

Figures 10A-10C provide quantitation of the amount of capsid protein present in a sample as measured in the dot blots shown in Figure 9, along with the amount of viral genomic DNA as measured by quantitative PCR and the detection of the pDISC-GFP gene and superimposed against the chromatogram for the separation of empty and full AAV6 (Figure 10A), AAV5 (Figure 10B), and AAV8 (Figure IOC) particles. Each chromatogram shows the absorption of the eluate and flow-through at 260 nm (purple or red line) and 280 nm (blue line) in milli Absorption Units (mAU). Also provided is the conductivity shown in orange and the concentration of Buffer B shown in green.

Figure 11 provides a chromatogram of the separation of empty and full AAV9 particles using magnesium chloride.

DETAILED DESCRIPTION OF THE INVENTION

1.1 _ References and Definitions

The patent and scientific literature referred to herein establishes knowledge that is available to those of skill in the art. The issued US patents, allowed applications, published foreign applications, and references, including GenBank database sequences, which are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference.

The present invention can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. For example, features illustrated with respect to one embodiment can be incorporated into other embodiments, and features illustrated with respect to a particular embodiment can be deleted from that embodiment. In addition, numerous variations and additions to the embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant invention.

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 invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference herein in their entirety.

As used herein, “a,” “an,” or “the” can mean one or more than one. For example, “a” cell can mean a single cell or a multiplicity of cells.

As used herein, unless specifically indicated otherwise, the word “or” is used in the inclusive sense of “and/or” and not the exclusive sense of “either/or.”

As used herein, an “adeno-associated viral particle” or “adeno-associated virus particle” or “AAV particle” refer to an adeno-associated capsid shell that may or may not comprise a viral genome encapsulated therein.

With respect to an AAV viral particle, the terms “empty” and “full” refer to an AAV particle lacking a viral genome and an AAV particle comprising a viral genome encapsulated therein, respectively. The presently disclosed methods can be utilized for the purification of full rAAV of any serotype. As used herein, the term “serotype” refers to a distinct variant within a species of virus that is determined based on the viral cell surface antigens. Known serotypes of AAV include, among others, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrhlO, and AAVrh74 (Weitzman and Linden (2011) In Snyder and Moullier Adeno-associated virus methods and protocols. Totowa, NJ: Humana Press).

The presently disclosed methods are useful for the purification of recombinant AAV. As used herein, with respect to a virus, the term “recombinant” means having an altered nucleic acid or amino acid sequence as compared to a native sequence as a result of the application of genetic engineering techniques.

As used herein, with respect to a protein, the term “recombinant” or “engineered” means having an altered amino acid sequence as compared to a native sequence as a result of the application of genetic engineering techniques to nucleic acids which encode the protein, and cells or organisms which express the protein. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation and other gene transfer technologies; homologous recombination; site-directed mutagenesis; and gene fusion. In accordance with this definition, a protein having an amino acid sequence identical to a naturally-occurring protein, but produced by cloning and expression in a heterologous host, is not considered recombinant.

As used herein, the term “separating” as it relates to empty and full rAAV particles, refers to the ability to produce two different eluate fractions from an anion exchange chromatography wherein the majority of AAV particles within a first eluate are empty AAV particles and the majority of AAV particles within a second eluate are full AAV particles. In some embodiments, the percentage of empty AAV particles within the first eluate is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or more. In particular embodiments, the percentage of full AAV particles within the second eluate is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or more. The percentage of empty or full AAV particles within a particular sample can be estimated using any method known in the art, but can be estimated using an immunoblot to detect capsid proteins, which can be quantitated by comparison to a standard curve of known amounts of virus (as described elsewhere herein) as compared to results of quantitative PCR using primers specific for viral genomic DNA or a gene of interest, as described elsewhere herein. Alternatively, the percentage of empty or full AAV particles within a particular sample can be determined using density gradient centrifugation.

As used herein, the term “viral sample” as it relates to the present invention refers to any type of sample comprising an AAV particle. The viral sample may be a host cell lysate that has been infected with or transduced by a virus or transfected with viral DNA. In other embodiments, the viral sample is an eluate of a host cell sample that has been affinity purified for AAV particles. In still other embodiments, the viral sample is a biological sample from a subject infected with a virus.

As used herein, “anion exchange chromatography” refers to a type of ion exchange chromatography in which ions and polar molecules are separated based on their affinity to the anion exchanger. Anion exchange chromatography utilizes an anion exchange chromatography medium that comprises a functionalized support that results in this stationary phase being positively charged. The mobile phase (i.e., the buffer comprising the viral sample) is passed over the stationary phase, allowing negatively charged molecules from the mobile phase to bind to the positively charged anion exchange chromatography medium. The anion exchange chromatography medium can be housed within a closed environment, such as a column. The anion exchange chromatography medium can be of any variety of packing style or chemical structure. The anion exchange chromatography medium can comprise particles (e.g., silica) compressed into a column. As used herein, a “monolithic column” refers to porous rod structures characterized by mesopores (pores with diameters between 2 and 50 nm) and macropores (pores with diameters of at least 50 nm in diameter) that provide the monolith with high permeability, a large number of channels, and a high surface area available for reactivity. Monolithic columns have pore connectivity values greater than 3, meaning that an analyte in a monolith is able to enter one channel and exit through any of 3 or more different venues.

As used herein, the term “quaternary amine” refers to a molecule having four organic substituents on a nitrogen atom, which are also referred to as quaternary ammonium cations with a positively charged nitrogen center.

As used herein, the term “loading” as it relates to chromatography refers to the process by which the mobile phase comprising a sample is passed over or through a chromatography medium such that molecules within the sample that are attracted to the chromatography medium are able to bind to the medium.

As used herein, the term “eluting” as it refers to chromatography refers to the process by which the attraction between the molecules from a sample and a chromatography medium is reduced such that the bound molecules are released.

As used herein, the term “elution gradient” refers to changes of the mobile phase composition during chromatography that results in the elution of bound molecules from the chromatography medium. The elution gradient may be linear with steady, gradual changes in the mobile phase, wherein increasing concentrations of a component is added to the mobile phase. Alternatively, the elution step may not comprise a gradient at all, but be an isocratic elution, wherein the mobile phase is kept constant. In other embodiments, the elution step comprises the use of an isocratic elution gradient or step gradient. As used herein, an “isocratic elution gradient” or “step gradient” refers to a gradient wherein the composition of the mobile phase is changed in steps during a single chromatographic run. In each step of an isocratic elution gradient, the mobile phase can be kept at the same composition until a subsequent step in the chromatographic run, at which time the composition of the mobile phase is changed. As used herein, the term “eluate” refers to a solution obtained by elution. As used herein, the term “conductivity” refers to the ability of a solution to conduct electricity. The SI unit of conductivity is siemens per meter (S/m).

As used herein, the term “affinity chromatography” refers to a method of separating a mixture of molecules based on their specific interaction with a binding partner and involves the use of a stationary phase comprising a binding partner of a molecule that is to be enriched and is present in the mobile phase that passes through or over the stationary phase. The affinity chromatography medium has affinity or specificity for a particular molecule(s).

Much like anion exchange chromatography medium, affinity chromatography medium can be housed within a closed environment, such as a column, and can be of any variety of packing style or chemical structure. The anion exchange chromatography medium can comprise particles (e.g., silica) compressed into a column, wherein the particles are functionalized with a binding partner having affinity for a molecule of interest.

The methods described herein rely on the use magnesium chloride as the sole salt to separate and elute empty rAAV particles and full rAAV particles within a viral sample from an anion exchange chromatography medium. Accordingly, isocratic and linear buffers utilized in the presently disclosed methods can comprise magnesium chloride, and do not comprise an additional salt, or additional salts, at a total concentration which affects separation of empty rAAV particles and full rAAV particles in the method of the invention.

In other words, if one or more additional salts are present, they do not result in an increase in the percentage of empty rAAV particles found in fractions containing full rAAV particles.

It is understood that the isocratic and linear buffers of the methods may comprise magnesium chloride and one or more additional salts, so long as the total concentration of the one or more additional salts is low enough that it does not affect rAAV particle separation in the method of the invention. Such additional salts can include, for example, sodium chloride at a total concentration that does not affect separation of empty rAAV particles and full rAAV particles in the method of the invention. Methods for determining concentrations at which different salts would affect separation of empty and full rAAV particles would be known to those of skill in the art and could further be determined by a skilled person using the methods described herein. It is also understood that the phrase “one or more additional salts at a total concentration which affects separation of empty rAAV particles and full rAAV particles” refers to the total or cumulative concentration of salts, other than magnesium chloride, present in the buffers, and that the concentration of such salts is sufficiently low that they do not affect the separation of empty rAAV particles from full rAAV particles in the method of the invention; i.e., they do not result in an increase in the percentage of empty rAAV particles in fractions containing full rAAV particles. It is further understood that the buffers of the invention may comprise magnesium chloride only, and no detectable concentration of another salt. The rAAV that are purified using the presently disclosed methods can express a gene of interest. In some embodiments, the gene of interest encodes a nuclease, such as an engineered nuclease. As used herein, the terms “nuclease” and “endonuclease” refers to enzymes which cleave a phosphodiester bond within a polynucleotide chain, such as a meganuclease, a transcription activator-like effector nuclease (TALEN), a compact TALEN, a zinc finger nuclease, a CRISPR system nuclease, or a megaTAL.

As used herein, the term “meganuclease” refers to an endonuclease that binds double- stranded DNA at a recognition sequence that is greater than 12 base pairs. Preferably, the recognition sequence for a meganuclease of the invention is 22 base pairs. A meganuclease can be, for example, an endonuclease that is derived from I-Crel, and can refer to an engineered variant of I-Crel that has been modified relative to natural I-Crel with respect to, for example, DNA-binding specificity, DNA cleavage activity, DNA-binding affinity, or dimerization properties. Methods for producing such modified variants of I-Crel are known in the art (e.g. WO 2007/047859). A meganuclease as used herein binds to double-stranded DNA as a heterodimer. A meganuclease may also be a “single-chain meganuclease” in which a pair of DNA-binding domains are joined into a single polypeptide using a peptide linker. The term “homing endonuclease” is synonymous with the term “meganuclease.” Meganucleases of the invention are substantially non-toxic when expressed in cells, such that cells can be transfected and maintained at 37oC without observing deleterious effects on cell viability or significant reductions in meganuclease cleavage activity when measured using the methods described herein.

As used herein, the term “single-chain meganuclease” refers to a polypeptide comprising a pair of meganuclease subunits joined by a linker. A single-chain meganuclease has the organization: N-terminal subunit - Linker - C-terminal subunit. The two meganuclease subunits will generally be non-identical in amino acid sequence and will recognize non-identical DNA sequences. Thus, single-chain meganucleases typically cleave pseudo-palindromic or non-palindromic recognition sequences. A single-chain meganuclease may be referred to as a “single-chain heterodimer” or “single-chain heterodimeric meganuclease” although it is not, in fact, dimeric. For clarity, unless otherwise specified, the term “meganuclease” can refer to a dimeric or single-chain meganuclease. As used herein, the term “linker” refers to an exogenous peptide sequence used to join two meganuclease subunits into a single polypeptide. A linker may have a sequence that is found in natural proteins, or may be an artificial sequence that is not found in any natural protein. A linker may be flexible and lacking in secondary structure or may have a propensity to form a specific three-dimensional structure under physiological conditions. A linker used in a single-chain meganuclease can include, without limitation, those encompassed by U.S. Patent Nos. 8,445,251, 9,340,777, 9,434,931, and 10,041,053.

As used herein, the term “TALEN” refers to an endonuclease comprising a DNA- binding domain comprising a plurality of TAL domain repeats fused to a nuclease domain or an active portion thereof from an endonuclease or exonuclease, including but not limited to a restriction endonuclease, homing endonuclease, S 1 nuclease, mung bean nuclease, pancreatic DNAse I, micrococcal nuclease, and yeast HO endonuclease. See, for example, Christian et al. (2010) Genetics 186:757-761, which is incorporated by reference in its entirety. Nuclease domains useful for the design of TALENs include those from a Type IIs restriction endonuclease, including but not limited to Fokl, FoM, Stsl, Hhal, Hindlll, Nod, BbvCI, EcoRI, Bgll, and AlwI. Additional Type IIs restriction endonucleases are described in International Publication No. WO 2007/014275. In some embodiments, the nuclease domain of the TALEN is a Fokl nuclease domain or an active portion thereof. TAL domain repeats can be derived from the TALE (transcription activator-like effector) family of proteins used in the infection process by plant pathogens of the Xanthomonas genus. TAL domain repeats are 33-34 amino acid sequences with divergent 12th and 13th amino acids. These two positions, referred to as the repeat variable dipeptide (RVD), are highly variable and show a strong correlation with specific nucleotide recognition. Each base pair in the DNA target sequence is contacted by a single TAL repeat, with the specificity resulting from the RVD.

In some embodiments, the TALEN comprises 16-22 TAL domain repeats. DNA cleavage by a TALEN requires two DNA recognition regions (i.e., “half-sites”) flanking a nonspecific central region (i.e., the “spacer”). The term “spacer” in reference to a TALEN refers to the nucleic acid sequence that separates the two nucleic acid sequences recognized and bound by each monomer constituting a TALEN. The TAL domain repeats can be native sequences from a naturally-occurring TALE protein or can be redesigned through rational or experimental means to produce a protein which binds to a pre-determined DNA sequence (see, for example, Boch et al. (2009) Science 326(5959): 1509- 1512 and Moscou and Bogdanove (2009) Science 326(5959): 1501, each of which is incorporated by reference in its entirety). See also, U.S. Publication No. 20110145940 and International Publication No. WO 2010/079430 for methods for engineering a TALEN to recognize a specific sequence and examples of RVDs and their corresponding target nucleotides. In some embodiments, each nuclease (e.g., Fokl) monomer can be fused to a TAL effector sequence that recognizes a different DNA sequence, and only when the two recognition sites are in close proximity do the inactive monomers come together to create a functional enzyme. It is understood that the term “TALEN” can refer to a single TALEN protein or, alternatively, a pair of TALEN proteins (i.e., a left TALEN protein and a right TALEN protein) which bind to the upstream and downstream half-sites adjacent to the TALEN spacer sequence and work in concert to generate a cleavage site within the spacer sequence. Given a predetermined DNA locus or spacer sequence, upstream and downstream half-sites can be identified using a number of programs known in the art (Kornel Labun; Tessa G. Montague; James A. Gagnon; Summer B. Thyme; Eivind Valen. (2016). CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Research; doi:10.1093/nar/gkw398; Tessa G. Montague; Jose M. Cruz; James A. Gagnon; George M. Church; Eivind Valen. (2014). CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res. 42. W401-W407). It is also understood that a TALEN recognition sequence can be defined as the DNA binding sequence (i.e., half-site) of a single TALEN protein or, alternatively, a DNA sequence comprising the upstream half-site, the spacer sequence, and the downstream half- site.

As used herein, the term “compact TALEN” refers to an endonuclease comprising a DNA-binding domain with one or more TAL domain repeats fused in any orientation to any portion of the I-Tevl homing endonuclease or any of the endonucleases listed in Table 2 in U.S. Application No. 20130117869 (which is incorporated by reference in its entirety), including but not limited to Mmel, EndA, Endl, I-Basl, I-TevII, I-TevIII, I-Twol, Mspl, Mval, NucA, and NucM. Compact TALENs do not require dimerization for DNA processing activity, alleviating the need for dual target sites with intervening DNA spacers. In some embodiments, the compact TALEN comprises 16-22 TAL domain repeats.

As used herein, the term “zinc finger nuclease” or “ZFN” refers to a chimeric protein comprising a zinc finger DNA-binding domain fused to a nuclease domain from an endonuclease or exonuclease, including but not limited to a restriction endonuclease, homing endonuclease, S 1 nuclease, mung bean nuclease, pancreatic DNAse I, micrococcal nuclease, and yeast HO endonuclease. Nuclease domains useful for the design of zinc finger nucleases include those from a Type IIs restriction endonuclease, including but not limited to Fokl, FoM, and Stsl restriction enzyme. Additional Type IIs restriction endonucleases are described in International Publication No. WO 2007/014275, which is incorporated by reference in its entirety. The structure of a zinc finger domain is stabilized through coordination of a zinc ion. DNA binding proteins comprising one or more zinc finger domains bind DNA in a sequence-specific manner. The zinc finger domain can be a native sequence or can be redesigned through rational or experimental means to produce a protein which binds to a pre-determined DNA sequence -18 basepairs in length, comprising a pair of nine basepair half-sites separated by 2-10 basepairs. See, for example, U.S. Pat. Nos. 5,789,538, 5,925,523, 6,007,988, 6,013,453, 6,200,759, and International Publication Nos. WO 95/19431, WO 96/06166, WO 98/53057, WO 98/54311, WO 00/27878, WO 01/60970, WO 01/88197, and WO 02/099084, each of which is incorporated by reference in its entirety. By fusing this engineered protein domain to a nuclease domain, such as Fokl nuclease, it is possible to target DNA breaks with genome-level specificity. The selection of target sites, zinc finger proteins and methods for design and construction of zinc finger nucleases are known to those of skill in the art and are described in detail in U.S. Publications Nos. 20030232410, 20050208489, 2005064474, 20050026157, 20060188987 and International Publication No. WO 07/014275, each of which is incorporated by reference in its entirety. In the case of a zinc finger, the DNA binding domains typically recognize an 18-bp recognition sequence comprising a pair of nine basepair “half-sites” separated by a 2-10 basepair “spacer sequence”, and cleavage by the nuclease creates a blunt end or a 5' overhang of variable length (frequently four basepairs). It is understood that the term “zinc finger nuclease” can refer to a single zinc finger protein or, alternatively, a pair of zinc finger proteins (i.e., a left ZFN protein and a right ZFN protein) which bind to the upstream and downstream half-sites adjacent to the zinc finger nuclease spacer sequence and work in concert to generate a cleavage site within the spacer sequence. Given a predetermined DNA locus or spacer sequence, upstream and downstream half-sites can be identified using a number of programs known in the art (Mandell JG, Barbas CF 3rd. Zinc Finger Tools: custom DNA-binding domains for transcription factors and nucleases. Nucleic Acids Res. 2006 Jul 1;34 (Web Server issue):W516-23). It is also understood that a zinc finger nuclease recognition sequence can be defined as the DNA binding sequence (i.e., half-site) of a single zinc finger nuclease protein or, alternatively, a DNA sequence comprising the upstream half-site, the spacer sequence, and the downstream half-site.

As used herein, the term “CRISPR” or “CRISPR system nuclease” refers to a system comprising a caspase-based endonuclease comprising a caspase, such as Cas9, and a guide RNA that directs DNA cleavage of the caspase by hybridizing to a recognition site in the genomic DNA. The caspase component of a CRISPR is an RNA-guided DNA endonuclease. In certain embodiments, the caspase is a class II Cas enzyme. In some of these embodiments, the caspase is a class II, type II enzyme, such as Cas9. In other embodiments, the caspase is a class II, type V enzyme, such as Cpfl. The guide RNA comprises a direct repeat and a guide sequence (often referred to as a spacer in the context of an endogenous CRISPR system), which is complementary to the target recognition site. In certain embodiments, the CRISPR further comprises a tracrRNA (trans-activating CRISPR RNA) that is complementary (fully or partially) to a direct repeat sequence (sometimes referred to as a tracr-mate sequence) present on the guide RNA. In particular embodiments, the caspase can be mutated with respect to a corresponding wild-type enzyme such that the enzyme lacks the ability to cleave one strand of a target polynucleotide, functioning as a nickase, cleaving only a single strand of the target DNA. Non-limiting examples of caspase enzymes that function as a nickase include Cas9 enzymes with a D10A mutation within the RuvC I catalytic domain, or with a H840A, N854A, or N863A mutation.

As used herein, the term “megaTAL” refers to a single-chain endonuclease comprising a transcription activator-like effector (TALE) DNA binding domain with an engineered, sequence-specific homing endonuclease.

In some embodiments, the gene of interest encodes a chimeric antigen receptor, an exogenous T cell receptor. As used herein, a “chimeric antigen receptor” or “CAR” refers to an engineered receptor that confers or grafts specificity for an antigen onto an immune effector cell (e.g., a human T cell). A chimeric antigen receptor typically comprises at least an extracellular ligand-binding domain or moiety and an intracellular domain that comprises one or more signaling domains and/or co-stimulatory domains.

In some embodiments, the extracellular ligand-binding domain or moiety is in the form of a single-chain variable fragment (scFv) derived from a monoclonal antibody, which provides specificity for a particular epitope or antigen (e.g., an epitope or antigen preferentially present on the surface of a cell, such as a cancer cell or other disease-causing cell or particle). In some embodiments, the scFv is attached via a linker sequence. In various embodiments, the extracellular ligand-binding domain is specific for any antigen or epitope of interest. In some embodiments, the scFv is murine, humanized, or fully human.

The extracellular domain of a chimeric antigen receptor can also comprise an autoantigen (see, Payne et al. (2016), Science 353 (6295): 179-184), that can be recognized by autoantigen- specific B cell receptors on B lymphocytes, thus directing T cells to specifically target and kill autoreactive B lymphocytes in antibody-mediated autoimmune diseases. Such CARs can be referred to as chimeric autoantibody receptors (CAARs), and their use is encompassed by the invention.

The extracellular domain of a chimeric antigen receptor can also comprise a naturally- occurring ligand for an antigen of interest, or a fragment of a naturally-occurring ligand which retains the ability to bind the antigen of interest.

The intracellular stimulatory domain can include one or more cytoplasmic signaling domains that transmit an activation signal to the immune effector cell following antigen binding. Such cytoplasmic signaling domains can include, without limitation, €ϋ3z. The intracellular stimulatory domain can also include one or more intracellular co- stimulatory domains that transmit a proliferative and/or cell-survival signal after ligand binding. Such intracellular co-stimulatory domains can be any of those known in the art and can include, without limitation, CD27, CD28, CD8, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and a ligand that specifically binds with CD83, Nl, N6, or any combination thereof.

A chimeric antigen receptor can further include additional structural elements, including a transmembrane domain that is attached to the extracellular ligand-binding domain via a hinge or spacer sequence. The transmembrane domain can be derived from any membrane-bound or transmembrane protein. For example, the transmembrane polypeptide can be a subunit of the T-cell receptor (i.e., an a, b, g or z, polypeptide constituting CD3 complex), IL2 receptor p55 (a chain), p75 (b chain) or g chain, subunit chain of Fc receptors (e.g., Fey receptor III) or CD proteins such as the CD8 alpha chain. Alternatively, the transmembrane domain can be synthetic and can comprise predominantly hydrophobic residues such as leucine and valine.

The hinge region refers to any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. For example, a hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. Hinge regions may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4 or CD28, or from all or part of an antibody constant region. Alternatively, the hinge region may be a synthetic sequence that corresponds to a naturally occurring hinge sequence, or may be an entirely synthetic hinge sequence. In particular examples, a hinge domain can comprise a part of a human CD8 alpha chain, FcyRllla receptor or IgGl. As used herein, an “exogenous T cell receptor” or “exogenous TCR” refers to a TCR whose sequence is introduced into the genome of an immune effector cell (e.g., a human T cell) that may or may not endogenously express the TCR. Expression of an exogenous TCR on an immune effector cell can confer specificity for a specific epitope or antigen (e.g., an epitope or antigen preferentially present on the surface of a cancer cell or other disease- causing cell or particle). Such exogenous T cell receptors can comprise alpha and beta chains or, alternatively, may comprise gamma and delta chains. Exogenous TCRs useful in the invention may have specificity to any antigen or epitope of interest.

As used herein, the recitation of a numerical range for a variable is intended to convey that the invention may be practiced with the variable equal to any of the values within that range. Thus, for a variable which is inherently discrete, the variable can be equal to any integer value within the numerical range, including the end-points of the range. Similarly, for a variable which is inherently continuous, the variable can be equal to any real value within the numerical range, including the end-points of the range. As an example, and without limitation, a variable which is described as having values between 0 and 2 can take the values 0, 1 or 2 if the variable is inherently discrete, and can take the values 0.0, 0.1, 0.01, 0.001, or any other real values between 0 and 2 if the variable is inherently continuous.

2.1 _ Principle of the Invention

The present invention is based, in part, on the discovery that magnesium chloride alone is sufficient for separating empty and full recombinant adeno-associated virus (rAAV) particles from a viral sample using an anion exchange chromatography medium and an isocratic elution gradient of the magnesium chloride. The use of a single salt in the elution step allows this method to be readily adaptable for the purification of full rAAV particles of any serotype. While not being bound by any theory or mechanism of action, it is believed that magnesium chloride is a suitable salt for these methods because it falls toward the chaotropic side of the Hoffmeister series, thus being able to destabilize weak interactions between molecules, and magnesium chloride itself has little immediate effect on AAV.

Thus, the present invention encompasses methods for effectively separating empty and full rAAV particles from viral samples. The present invention also encompasses viral compositions comprising full rAAV particles produced by the presently disclosed methods and pharmaceutical compositions comprising the viral compositions and a pharmaceutically acceptable carrier. Further, the invention encompasses methods for delivering a gene of interest by contacting a cell with the viral compositions or pharmaceutical compositions wherein the rAAV particle comprises the gene of interest.

2.2 _ Methods for Separating Empty and Full Recombinant Adeno- Associated Viral

Particles

The present disclosure provides methods for separating empty and full rAAV particles from a viral sample by separately eluting empty and full rAAV particles using an isocratic elution gradient of magnesium chloride as the sole salt from an anion exchange chromatography medium that has been loaded with the viral sample.

The anion exchange chromatography medium comprises the stationary phase of anion exchange chromatography over which the mobile phase (i.e., the buffer comprising the viral sample) is passed. Typically, the anion exchange chromatography medium can be housed within a closed environment, such as a column. The anion exchange chromatography medium can be of any variety of packing style or chemical structure and can be functionalized.

Traditionally, anion exchange chromatography medium comprises particles, usually silica, compressed into a column. Given that back pressure of a column is inversely proportional to the square of the particle size and particle size is often decreased to achieve smaller diffusion distances and reduce run times and increase selectivity, monoliths are often used, particularly for large molecules. Thus, in some embodiments, the anion exchange chromatography medium comprises a monolithic column (i.e., a monolith), which exhibit short diffusion distances and less back pressure. Monoliths are cast as a single block and inserted into a chromatographic housing and are characterized by a highly interconnected network of channels. Binding sites are situated inside the channels and there are no diffusion limitations to monoliths, with performance being the same at lower and higher flow rates.

The channels of monoliths are generally large (1-2 pm), which is optimal for molecules like viruses.

While any anion exchange chromatography monolithic column can be used in the presently disclosed methods, in some embodiments, the anion exchange chromatography medium comprises a monolithic column comprising methacrylate, such as poly(glycidyl methacrylate-co-ethylene dimethacrylate) that makes up CIM^® media (commercially available from BIA Separations (Ajdovscina, Slovenia)).

The anion exchange chromatography medium comprises a positively charged functional group (i.e., chemistry) that aids in anion exchange. In some of these embodiments, the functional group that aids in anion exchange is a quaternary amine, which is a strong anion exchanger. In particular embodiments, the anion exchange chromatography medium comprises the quaternary amine of CIMmultus™ QA or CIM^® QA that is commercially available from BIA Separations. In some embodiments, the anion exchange chromatography medium is housed within a disposable housing comprised of an epoxy thermoset composite reinforced with carbon fibers and coated with parylene C, such as the housing of CIMmultus™ columns that are commercially available from BIA Separations. In some of these embodiments, the anion exchange chromatography medium comprises CIMmultus™ QA or CIM^® QA. CIM^® QA and CIMmultus™ QA columns are available in a 1 ml, 4 ml, 8 ml, 40 ml, 80 ml, 400 ml, 800 ml, 4000 ml, or 8000 ml column and the larger columns are more suitable for larger scale purification of full rAAV particles.

Generally, the steps of anion exchange chromatography involve equilibrating the anion exchange medium with buffer, preparing the sample (e.g., viral sample) by diluting the sample in the buffer (e.g., by buffer exchange), loading the sample onto the anion exchange medium, washing the anion exchange medium to remove any unbound fraction, and eluting the bound fraction using an elution gradient.

In some embodiments, the same buffer is used to equilibrate the anion exchange medium, prepare the sample, load the sample, and wash the anion exchange medium. The same buffer, with the addition of a salt (i.e., magnesium chloride), can also be used for elution of the bound fraction.

In certain embodiments of the presently disclosed methods, preparing the viral sample involves increasing the pH of the viral sample to between 8 and 9 with Bis-Tris propane. In some of these embodiments, the pH of the viral sample is increased to about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or any other value between 8 and 9. In certain embodiments, the pH of the viral sample is increased to about 8.5 with Bis-Tris propane.

While any type of aqueous buffer can be used in the presently disclosed methods for the equilibration, loading, and/or washing steps, in some embodiments, the buffer comprises Bis-Tris propane (i.e., 1,3-bispropane). Bis-Tris propane has a wide buffering range, from 6 to 9.5 due to is two pK_a values which are close in value. In some of those embodiments wherein the aqueous buffer used in the equilibration step, loading step, washing step, and/or elution step comprises Bis-Tris propane, the pH of the buffer is between 8 and 9. In certain embodiments, the pH of the aqueous buffer comprising Bis-Tris propane is about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or any other value between 8 and 9. In certain embodiments, the pH of the aqueous buffer comprising Bis-Tris propane is about 9, about 9.1, about 9.2, about 9.3, about 9.4, or about 9.5, or any other value between 9 and 9.5. In some of those embodiments wherein the aqueous buffer used in the equilibration step, loading step, washing step, and/or elution step comprises Bis-Tris propane, the concentration of Bis-Tris propane in the aqueous buffer is between 5 mM and 30 mM, including but not limited to about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, and any other value between 5 mM and 30 mM.

The loading step of anion exchange chromatography comprises adding the sample (e.g., viral sample), which can be prepared by dilution in an aqueous buffer (e.g., Bis-Tris propane) as described above, onto an equilibrated anion exchange chromatography medium and allowing the sample to flow through the anion exchange chromatography medium at such a flow rate that allows for the functionalized and positively charged anion exchange chromatography medium to bind negatively charged molecules. Given that the average pi of a full AAV particle is about 5.9 and the average pi of an empty AAV particle is about 6.3, the pH of the loading buffer should be at a pH above 6.3 to impart a negative charge onto the full and empty AAV particles, allowing them to bind to the positively charged anion exchange chromatography medium.

After the complete viral sample has been loaded onto the anion exchange chromatography medium, the medium is washed with an aqueous buffer, such as Bis-Tris propane, to remove the unbound fraction of the viral sample.

Following the wash step, the bound fraction of the viral sample is eluted using either a linear or isocratic (i.e., step-wise) gradient comprising a salt (i.e., magnesium chloride). The negative ions in the salt solution compete with the bound molecules in binding to the resin and the point within the gradient at which a bound molecule is eluted from the anion exchange chromatography medium is based on the overall charge of the molecule. Given that the average pi of a full AAV particle is about 5.9 and the average pi of an empty AAV particle is about 6.3, the empty AAV particle will elute prior to the full AAV particle when using anion exchange chromatography.

In some anion exchange chromatography protocols, more than one type of salt is needed to separately elute molecules (e.g., empty and full AAV particles). In the presently disclosed methods, however, magnesium chloride is sufficient to allow for the separate elution of empty and full AAV particles. The use of a single salt in the elution step allows for the method to be easily adapted to other AAV serotypes given that a particular conductivity reading can be readily translated into a concentration of magnesium chloride. In general, the salt concentration of magnesium chloride is gradually increased in a linear elution gradient by increasing the percentage or ratio of the elution buffer (comprising the magnesium chloride) to the loading/wash buffer (buffer only without magnesium chloride). In certain embodiments, the elution buffer comprises about 20 mM Bis-Tris propane at a pH of about 8.5 and magnesium chloride at a concentration between 1 mM and 100 mM, including but not limited to about 1 mM, about 2 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, or any other value between 1 mM and 100 mM. In some embodiments, the concentration of magnesium chloride within the elution buffer is between 20 mM and 60 mM, and in particular embodiments, the concentration of magnesium chloride within the elution buffer is about 40 mM. In some of these embodiments, the elution buffer comprises about 20 mM Bis-Tris propane at a pH of about 8.5 and about 40 mM magnesium chloride.

In order to identify the conductivity points at which the empty and full rAAV particles eluted to use in the isocratic elution gradient, an initial anion exchange chromatography step can be performed with a linear elution gradient of a fraction of the viral sample prior to the larger scale anion exchange chromatography with the isocratic elution gradient to identify the peaks in the chromatogram that correspond to empty and full rAAV particles. Using an isocratic elution gradient provides for more reproducible results, a shorter run time, and greater separation of eluted molecules.

The first step of the step-wise isocratic elution gradient corresponds to the elution of empty rAAV particles. This first step of the isocratic elution gradient can be identified as the conductivity (measured in milliSiemens/centimeter) at which absorbance of ultraviolet light at a wavelength of 280 nm (measured in milli Absorption units), which is indicative of protein, is greater than absorbance of UV light at a wavelength of 260 nm, which is indicative of nucleic acids, and absorbance at 280 nm is at or near its peak in the linear gradient. In some embodiments, the first step of the isocratic elution gradient is set as the conductivity wherein absorbance at 280 nm is greater than absorbance at 260, and absorbance at 280 nm is at its peak plus or minus of up to 0.5 mS/cm, including but not limited to about 0.05 mS/cm, about 0.1 mS/cm, about 0.15 mS/cm, about 0.2 mS/cm, about 0.25 mS/cm, about 0.3 mS/cm, about 0.35 mS/cm, about 0.4 mS/cm, about 0.45 mS/cm, and about 0.5 mS/cm. The second step of the isocratic elution gradient corresponds to the elution of full rAAV particles and can be identified as the conductivity at which absorbance at 260 nm is greater than absorbance at 280 nm, and absorbance at 260 nm is at or near its peak in the linear gradient. In some embodiments, the second step of the isocratic elution gradient is set as the conductivity wherein absorbance at 260 nm is greater than absorbance at 280 nm, and absorbance at 260 nm is at its peak plus or minus of up to 0.5 mS/cm, including but not limited to about 0.05 mS/cm, about 0.1 mS/cm, about 0.15 mS/cm, about 0.2 mS/cm, about 0.25 mS/cm, about 0.3 mS/cm, about 0.35 mS/cm, about 0.4 mS/cm, about 0.45 mS/cm, and about 0.5 mS/cm.

The percentage of the elution buffer that translates to the conductivities of the first and second step of the isocratic elution gradient can be determined using any method known in the art. In some embodiments, running a chromatographic system in the absence of an anion exchange medium (e.g., column) will allow for empirically determining the percentage of the elution buffer that results in a particular conductivity.

The process of performing a previous smaller-scale anion exchange chromatography with a fraction of a viral sample and a linear elution gradient can be repeated for each AAV serotype in order to determine the particular conductivities to use for the isocratic elution gradient for that particular serotype.

The presently disclosed methods can thus be used for any AAV serotype, naturally- occurring or synthetic, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV 6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrhlO, and AAVrh74. In some embodiments, the presently disclosed methods are used to separate empty and full AAV particles of serotype AAV5, AAV6, AAV8, or AAV9 (i.e., serotypes 5, 6, 8, or 9). Thus, in some embodiments of the disclosed methods, the rAAV comprises an rAAV5 particle, an rAAV6 particle, an rAAV8 particle, or an rAAV9 particle.

In some embodiments, a viral sample (e.g., host cell lysate) is enriched for AAV particles using affinity chromatography prior to anion exchange chromatography. Affinity chromatography involves the use of a stationary phase comprising a binding partner of a molecule that is to be enriched and is present in the mobile phase that passes through the stationary phase. In some embodiments, affinity chromatography takes advantage of the highly specific interaction between an enzyme and substrate, receptor and ligand, or antigen and antibody. In those embodiments of the presently disclosed methods in which the viral sample is enriched for AAV particles using affinity chromatography, the affinity chromatography medium has specificity for AAV. This specificity can be imparted by the use of an antibody or antigen-binding fragment thereof that specifically binds to a surface protein of AAV (e.g., capsid protein). Any affinity chromatography medium known in the art that has specificity for AAV can be used in the presently disclosed methods. Non-limiting examples of suitable affinity chromatography medium include AVB Sepharose™ that is available from GE Healthcare Life Sciences, POROS™ Captures elect™ AAV8, POROS™ CaptureSelect™ AAV9, and POROS™ CaptureSelect™ AAV-X, each of which is available from Thermo Fisher Scientific. AVB Sepharose™ affinity chromatography medium comprises a highly cross-linked 6% agarose matrix with an attached 14KD recombinant protein ligand from a single chain antibody that has affinity for AAV serotypes 1, 2, 3, and 5. The POROS™ CaptureSelect™ affinity chromatography medium is comprised of 50-pm, rigid, polymeric resin backbone comprised of crosslinked poly[styrene divinylbenzne] with a surface coated with a cross-linked polyhydroxylated polymer that is derivatized with an affinity ligand. The affinity ligand of the POROS™ CaptureSelect™ AAV8, AAV9, and AAV-X is a single-domain monospecific 13-kDa antibody fragment comprising the three complementarity determining regions (CDRs) that form the antigen-binding domain based on camelid-derived single-domain antibody fragments. The POROS™ CaptureSelect™ AAV-X affinity chromatography medium has demonstrated binding reactivity towards certain AAV serotypes, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAVrhlO. The POROS™ CaptureSelect™ AAV8 affinity chromatography medium has affinity for the AAV8 serotype and the POROS™ CaptureSelect™ AAV9 affinity chromatography medium has affinity for the AAV9 serotype. In certain embodiments, the affinity chromatography medium that is used in the affinity chromatography step of the presently disclosed methods is POROS™ CaptureSelect™ AAV-X.

The viral sample (e.g., host cell lysate) is loaded onto the affinity chromatography medium in a suitable buffer to allow for specific binding of AAV to the affinity chromatography medium, followed by a wash step to remove unbound molecules. In order to elute the bound AAV particles from the affinity chromatography medium, a low pH elution buffer is used and the eluate is collected. In some embodiments, the low pH elution buffer has a pH between 2 and 3. Any suitable buffer that buffers well at low pH may be used for the elution step, including but not limited to, phosphate, hydrochloric acid, glycine, acetate, propylene glycol, and citric acid.

In those embodiments wherein an affinity chromatography step precedes the anion exchange chromatography, the viral sample that is loaded onto the anion exchange chromatography medium is an enriched eluate from the affinity chromatography step that is enriched for rAAV particles. In some of these embodiments, the eluate from the affinity chromatography step is diluted in anion exchange loading buffer prior to loading the eluate onto the anion exchange chromatography medium.

Various flow rates and volumes of equilibration/loading/wash/elution buffer can be used in each of the steps of affinity chromatography and anion exchange chromatography. One of ordinary skill in the art of chromatography would understand how to vary the flow rate and volume of the buffer and/or sample when equilibrating, loading, washing, and eluting to maximize the efficacy of each step.

The eluate from affinity chromatography, anion exchange chromatography, or both can be analyzed for the presence of AAV particles by performing an immunoblot using an antibody or antigen-binding fragment thereof that recognizes an AAV surface protein (e.g., capsid protein). In certain embodiments, the antibody used in an immunoblot recognizes each of AAV VP1, VP2, and VP3 capsid proteins, such as clone B1 from LSBio (cat. no. C193393).

Immunoblotting does not distinguish empty from full AAV particles. In order to analyze eluate from affinity chromatography, anion exchange chromatography, or both for full AAV particles, quantitative PCR can be performed using primers that are specific for the viral genome or the gene of interest. The eluate sample can be prepared for quantitative PCR using any method known in the art for DNA extraction. In some embodiments, the eluate is first digested with DNase to remove non-viral or unpackaged DNA. In certain embodiments, the packaged DNA can be released by treating the eluate with Proteinase K.

Viral samples from which full rAAV particles are purified can comprise host cell lysate from cells that produce recombinant AAV, such as those that have been transfected with AAV vector(s), or the eluate from an affinity chromatography of a host cell lysate. Recombinant AAV are typically produced in mammalian cell lines such as HEK-293, however, any other suitable host cell known in the art may be used. Because the viral cap and rep genes are removed from the recombinant AAV vector to prevent its self-replication to make room for the gene(s) of interest to be delivered (e.g., a nuclease gene or chimeric antigen receptor gene), it is necessary to provide these in trans in the packaging cell line. In addition, it is necessary to provide the “helper” (e.g., adenoviral) components necessary to support replication (Cots D, Bosch A, Chillon M (2013) Curr. Gene Ther. 13(5): 370-81). Frequently, recombinant AAV are produced using a triple-transfection in which a cell line is transfected with a first plasmid encoding the “helper” components, a second plasmid comprising the cap and rep genes, and a third plasmid comprising the viral inverted terminal repeats (ITRs) flanking the intervening gene of interest sequence to be packaged into the vims. Viral particles are then isolated from cells by freeze-thaw cycles, sonication, detergent, or other means known in the art to produce a host cell lysate.

Because recombinant AAV particles are typically produced (manufactured) in cells, in those embodiments wherein the gene of interest is a nuclease, precautions must be taken in practicing the current invention to ensure that the nuclease is not expressed in the packaging cells. Because the viral genomes of the invention may comprise a recognition sequence for the nuclease, any nuclease expressed in the packaging cell line may be capable of cleaving the viral genome before it can be packaged into viral particles. This will result in reduced packaging efficiency and/or the packaging of fragmented genomes. Several approaches can be used to prevent nuclease expression in the packaging cells, including:

1. The nuclease can be placed under the control of a tissue-specific promoter that is not active in the packaging cells. For example, if a viral vector is developed for delivery of (a) nuclease gene(s) to muscle tissue, a muscle- specific promoter can be used. Examples of muscle-specific promoters include C5-12 (Liu, et al. (2004) Hum Gene Ther. 15:783-92), the muscle-specific creatine kinase (MCK) promoter (Yuasa, et al. (2002) Gene Ther. 9:1576-88), or the smooth muscle 22 (SM22) promoter (Haase, et al. (2013) BMC Biotechnol. 13:49-54). Examples of CNS (neuron) -specific promoters include the NSE, Synapsin, and MeCP2 promoters (Lentz, et al. (2012) Neurobiol Dis. 48:179-88). Examples of liver- specific promoters include albumin promoters (such as Palb), human aΐ-antitrypsin (such as PalAT), and hemopexin (such as Phpx) (Kramer, MG et al., (2003) Mol. Therapy 7:375-85). Examples of eye-specific promoters include opsin, and comeal epithelium- specific K12 promoters (Martin KRG, Klein RL, and Quigley HA (2002) Methods (28): 267-75) (Tong Y, et al., (2007) / Gene Med , 9:956-66). These promoters, or other tissue-specific promoters known in the art, are not highly-active in HEK-293 cells and, thus, will not be expected to yield significant levels of nuclease gene expression in packaging cells when incorporated into viral vectors. Similarly, the methods of the present invention contemplate the use of other host cell lines with the use of incompatible tissue specific promoters (i.e., the well-known HeLa cell line (human epithelial cell) and using the liver- specific hemopexin promoter). Other examples of tissue specific promoters include: synovial sarcomas PDZD4 (cerebellum), C6 (liver), ASB5 (muscle), PPP1R12B (heart), SLC5A12 (kidney), cholesterol regulation APOM (liver), ADPRHL1 (heart), and monogenic malformation syndromes TP73L (muscle). (Jacox E, et al., (2010) PLoS One v.5(8):el2274). Alternatively, the vector can be packaged in cells from a different species in which the nuclease is not likely to be expressed. For example, viral particles can be produced in microbial, insect, or plant cells using mammalian promoters, such as the well-known cytomegalovirus- or SV40 virus-early promoters, which are not active in the non-mammalian packaging cells. In a preferred embodiment, viral particles are produced in insect cells using the baculovirus system as described by Gao, et al. (Gao, H., et al. (2007) J. Biotechnol. 131(2): 138-43). A nuclease under the control of a mammalian promoter is unlikely to be expressed in these cells (Airenne, KJ, et al. (2013) Mol. Ther. 21(4):739-49). Moreover, insect cells utilize different mRNA splicing motifs than mammalian cells. Thus, it is possible to incorporate a mammalian intron, such as the human growth hormone (HGH) intron or the SV40 large T antigen intron, into the coding sequence of a nuclease. Because these introns are not spliced efficiently from pre-mRNA transcripts in insect cells, insect cells will not express a functional nuclease and will package the full-length genome. In contrast, mammalian cells to which the resulting recombinant AAV particles are delivered will properly splice the pre-mRNA and will express functional nuclease protein. Haifeng Chen has reported the use of the HGH and SV40 large T antigen introns to attenuate expression of the toxic proteins barnase and diphtheria toxin fragment A in insect packaging cells, enabling the production of recombinant AAV vectors carrying these toxin genes (Chen, H (2012) Mol Ther Nucleic Acids. 1(11): e57). The nuclease gene can be operably linked to an inducible promoter such that a small-molecule inducer is required for nuclease expression. Examples of inducible promoters include the Tet-On system (Clontech; Chen H., et al., (2015) BMC Biotechnol. 15(1):4)) and the RheoSwitch system (Intrexon; Sowa G., et al.,

(2011) Spine, 36(10): E623-8). Both systems, as well as similar systems known in the art, rely on ligand-inducible transcription factors (variants of the Tet Repressor and Ecdysone receptor, respectively) that activate transcription in response to a small-molecule activator (Doxycycline or Ecdysone, respectively). Practicing the current invention using such ligand-inducible transcription activators includes: 1) placing the nuclease gene under the control of a promoter that responds to the corresponding transcription factor, the nuclease gene having (a) binding site(s) for the transcription factor; and 2) including the gene encoding the transcription factor in the packaged viral genome The latter step is necessary because the nuclease will not be expressed in the target cells or tissues following recombinant AAV delivery if the transcription activator is not also provided to the same cells. The transcription activator then induces nuclease gene expression only in cells or tissues that are treated with the cognate small-molecule activator. This approach is advantageous because it enables nuclease gene expression to be regulated in a spatio-temporal manner by selecting when and to which tissues the small- molecule inducer is delivered. However, the requirement to include the inducer in the viral genome, which has significantly limited carrying capacity, creates a drawback to this approach. In another preferred embodiment, recombinant AAV particles are produced in a mammalian cell line that expresses a transcription repressor that prevents expression of the nuclease. Transcription repressors are known in the art and include the Tet-Repressor, the Lac-Repressor, the Cro repressor, and the Lambda- repressor. Many nuclear hormone receptors such as the ecdysone receptor also act as transcription repressors in the absence of their cognate hormone ligand. To practice the current invention, packaging cells are transfected/transduced with a vector encoding a transcription repressor and the nuclease gene in the viral genome (packaging vector) is operably linked to a promoter that is modified to comprise binding sites for the repressor such that the repressor silences the promoter. The gene encoding the transcription repressor can be placed in a variety of positions. It can be encoded on a separate vector; it can be incorporated into the packaging vector outside of the ITR sequences; it can be incorporated into the cap/rep vector or the adenoviral helper vector; or it can be stably integrated into the genome of the packaging cell such that it is expressed constitutively. Methods to modify common mammalian promoters to incorporate transcription repressor sites are known in the art. For example, Chang and Roninson modified the strong, constitutive CMV and RSV promoters to comprise operators for the Lac repressor and showed that gene expression from the modified promoters was greatly attenuated in cells expressing the repressor (Chang BD, and Roninson IB (1996) Gene 183:137-42). The use of a non-human transcription repressor ensures that transcription of the nuclease gene will be repressed only in the packaging cells expressing the repressor and not in target cells or tissues transduced with the resulting recombinant AAV vector.

2.3 Viral Compositions and Pharmaceutical Compositions

In some aspects, the present disclosure provides a viral composition produced by the presently disclosed methods in which the sample has been enriched for full rAAV particles. In some embodiments, the viral composition comprises a higher ratio of full rAAV particles to empty rAAV particles when compared to a viral composition produced using any other method known in the art for purification of full rAAV particles.

In certain aspects, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a viral composition produced by the presently disclosed methods of separating empty and full rAAV particles. Pharmaceutical compositions of the invention can be useful for treating a subject in need of gene therapy or gene editing.

Such pharmaceutical compositions can be prepared in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (21^st ed. 2005). In the manufacture of a pharmaceutical formulation according to the invention, viral compositions are typically admixed with a pharmaceutically acceptable carrier and the resulting composition is administered to a subject. The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the subject. In some embodiments, pharmaceutical compositions of the invention can further comprise one or more additional agents or biological molecules useful in the treatment of a disease in the subject. Likewise, the additional agent(s) and/or biological molecule(s) can be co-administered as a separate composition.

2.4 Methods for Delivering a Gene of Interest

In some aspects, the present disclosure provides methods for delivering a gene of interest, wherein the method comprises contacting a cell with a viral composition produced by the presently disclosed methods or a pharmaceutical composition comprising the same, wherein the full rAAV particles comprise the gene of interest. In some embodiments, the gene of interest encodes an engineered nuclease, such as an engineered meganuclease, a TALEN, a compact TALEN, a zinc finger nuclease, a CRISPR system nuclease, or a megaTAL. In other embodiments, the gene of interest encodes a chimeric antigen receptor, exogenous T cell receptor, or other chimeric antigen binding molecule. The cell or population thereof is contacted with the viral composition or pharmaceutical composition comprising the same such that the rAAV particles are able to infect the cell or a fraction of the population of cells. Given that AAV infect both dividing and non-dividing cells, the cells that can be contacted with the presently disclosed viral or pharmaceutical compositions need not be cycling. In some embodiments, the cells that are contacted for delivery of the gene of interest can include T cells. In some such embodiments, the rAAV particles comprise a gene encoding a chimeric antigen receptor, exogenous T cell receptor, or other chimeric antigen receptor. In further embodiments, the rAAV particles can be contacted with any cell type of interest, whether in vivo or ex vivo. Those of skill in the art would understand and select the appropriate rAAV serotype for delivery of a gene to a specific cell or tissue type.

Following infection of the cell or population thereof with the viral composition comprising full rAAV particles, the cell(s) can be cultured in such a manner as to allow expression of the gene of interest.

The presently disclosed methods for delivering a gene of interest can occur in an in vitro or in vivo setting. In those embodiments wherein the presently disclosed viral compositions or pharmaceutical compositions thereof are used to deliver a gene of interest to a cell (e.g., T cell) in vitro, following infection by the viral composition, the cell (e.g., T cell) can be delivered to a patient as an allogeneic or autologous therapy.

In one embodiment, T cells which have been removed from a subject can be contacted with a viral composition produced by the presently disclosed methods. The infected T cells which have been removed from the subject can then be returned to the same subject, or to a different subject, and conditions are provided which are conducive to expression of the gene of interest.

For those embodiments in which it is desired to express the gene of interest that has been delivered to a cell, the gene of interest can be operably linked to a promoter to facilitate transcription of the gene of interest. Mammalian promoters suitable for the invention include constitutive promoters such as the cytomegalovirus early (CMV) promoter (Thomsen et al. (1984), Proc Natl Acad Sci USA. 81(3):659-63) or the SV40 early promoter (Benoist and Chambon (1981), Nature. 290(5804):304-10) as well as inducible promoters such as the tetracycline-inducible promoter (Dingermann et al. (1992), Mol Cell Biol. 12(9):4038-45). The gene of interest can also be operably linked to a synthetic promoter. Synthetic promoters can include, without limitation, the JeT promoter (WO 2002/012514). In specific embodiments, the gene of interest is comprised within an expression cassette (i.e., “cassette”) comprising a promoter and the gene of interest. In other embodiments, the full rAAV particles comprise at least a first cassette and a second cassette.

In other embodiments, the full rAAV particles comprise a cassette comprising a promoter and a polycistronic nucleic acid sequence, wherein the promoter drives expression of the polycistronic nucleic acid sequence to generate a polycistronic mRNA in an infected cell, in which the polycistronic nucleic acid sequence comprises more than one gene of interest. A polycistronic mRNA of the invention can comprise any element known in the art to allow for the translation of two or more genes from the same mRNA molecule including, but not limited to, an IRES peptide, a T2A peptide, a P2A peptide, an E2A peptide, and an F2A peptide.

2.5 Methods for Treating Diseases

Disclosed herein are methods for treating a disease associated with a gene of interest such that expression of the gene of interest treats the disease. The present disclosure thus provides a method for treating a disease associated with a gene of interest, wherein the method comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a viral composition produced by the presently disclosed methods, wherein expression of the gene of interest treats the disease.

The viral compositions produced by the presently disclosed methods of affinity chromatography with an isocratic elution gradient of magnesium chloride are enriched for full rAAV particles and thus are more efficacious than viral compositions in which the presently disclosed affinity chromatography purification has not been performed. The high percentage of full rAAV particles within the presently disclosed viral compositions is also expected to improve the safety profile of the viral composition upon administration to a subject given that less empty rAAV particles are present within the viral composition to elicit responses by the subject’s immune system.

In one embodiment, wherein the viral composition comprises a gene encoding an engineered nuclease, the virus comprises a self-limiting virus. A self-limiting vims can have limited persistence time in a cell or organism due to the presence of a recognition sequence for an engineered nuclease within the viral vector. Thus, a self-limiting virus can be engineered to provide coding for a promoter, an engineered nuclease, and a nuclease recognition site within the ITRs. The self-limiting vims delivers the nuclease gene to a cell, tissue, or organism, such that the nuclease is expressed and able to cut the genome of the cell at an endogenous recognition sequence within the genome. The delivered nuclease will also find its target site within the self-limiting virus itself, and cut the vector at this target site. Once cut, the 5' and 3' ends of the viral genome will be exposed and degraded by exonucleases, thus killing the virus and ceasing production of the nuclease.

In some embodiments, the viral or pharmaceutical compositions are injected directly into target tissues. In alternative embodiments, the viral or pharmaceutical compositions are delivered systemically via the circulatory system. It is known in the art that different AAV serotypes tend to localize to different tissues.

Appropriate doses will depend, among other factors, on the specific serotype of AAV, on the route of administration, on the subject being treated (i.e., age, weight, sex, and general condition of the subject), and the mode of administration. Thus, the appropriate dosage may vary from patient to patient. An appropriate effective amount can be readily determined by one of skill in the art. Dosage treatment may be a single dose schedule or a multiple dose schedule. Moreover, the subject may be administered as many doses as appropriate. One of skill in the art can readily determine an appropriate number of doses. The dosage may need to be adjusted to take into consideration an alternative route of administration, or balance the therapeutic benefit against any side effects.

EXAMPLES

This invention is further illustrated by the following examples, which should not be construed as limiting. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are intended to be encompassed in the scope of the claims that follow the examples below.

EXAMPLE 1

Attempted separation of empty and full AAV6 particles using the CIMmultus™ QA monolith with sodium chloride elution

Initial attempts to separate empty and full particles of AAV6 using the CIMmultus™ QA column were performed using sodium chloride in the elution gradient. As shown in Figure 1, the gradient was not able to distinctly separate empty from full particles even though there was a slight UV260/280 cross-over event indicated by the hashed line.

Although slight separation was indeed observable, the distinction between the two peaks (i.e., empty and full particles) was not sufficient enough to delineate a clear fraction that did not contain empty particles.

EXAMPLE 2

Attempted separation of empty and full AAV6 particles using the CIMmultus™ QA monolith with pH gradient elution

Although not all too common, in some instances, a pH gradient can be used to elute proteins bound to an anion exchanger. In this case, the pH gradient must be from high to low. As shown in Figure 2, the trace remained close to baseline with no visible elution of protein from the column. Thus, pH was not a feasible means of separation for this purpose.

EXAMPLE 3

Separation of empty and full AAV6 particles using the CIMmultus QA monolith with magnesium chloride elution

As depicted in Figure 3, magnesium chloride was able to elicit the separation crossover event (shown in the circled area of the chromatogram). This was a direct indication that MgCh alone was able to separate empty from full particles.

The visible indication of the crossover event led to the broadening of the gradient specifically to isolate the point where the crossover occurs. The resolution depicted by the crossover event was not broad enough to efficiently resolve empty particles from full particles. Thus, to further tease apart the two species, the gradient was further shallowed as depicted in Figure 4. The arrows indicate the conductivity that was targeted in the subsequent run. This allowed further focusing of the gradient, allowing even better resolution of the two peaks (Figure 5). The arrows indicate the conductivity that was targeted for the isocratic elution in Example 4.

EXAMPLE 4

Separation of empty and full AAV6 using the CIMmultus™ QA monolith with magnesium chloride in an isocratic gradient

Being able to see distinct peaks in the chromatogram in Example 3 allowed choosing distinct points in the conductivity (mS) trace, which further allowed the transformation of the process from a linear gradient to an isocratic elution. By running the system without a column in place, these distinct conductivities could be pinpointed by adjusting the percentage of Buffer A ( 20mM Bis-Tris propane pH 8.5) to Buffer B (20mM Bis-Tris propane pH 8.5, 40mM MgC12). The percentages were as follows: wash at 0% Buffer B, elution of empty particles at 58.5% Buffer B, elution of full particles at 80% Buffer B, and elution of aggregates at 100% Buffer B. The corresponding specific conductivities for these percentages of Buffer B were 6.2 mS/cm, 7.8 mS/cm, and 9.2 mS/cm for elution of empty particles, elution of full particles, and elution of aggregates, respectively. The isocratic gradient is shown in Figure 6A. To determine if this method was repeatable, two subsequent preps were purified using the same method (Figures 6B and 6C).

EXAMPLE 5

Separation of empty and full AAV5 and AAV8 using the CIMmultus™ QA monolith with magnesium chloride in an isocratic gradient

Following the success of the experiments in Example 4, the application of the same approach to other AAV serotypes was tested. Self-complementary AAV5 vector was generated using the triple transfection approach and purified using affinity chromatography. The affinity peak was then diluted and loaded onto the CIMmultus™ QA column and eluted using a linear gradient. Using the same approach as Example 4, the gradient was shallowed out to find specific conductivities that would allow for an isocratic gradient. The results are shown in Figure 7 and the percentages were as follows: wash at 0% Buffer B, elution of empty particles at 50.0% Buffer B, elution of full particles at 61% Buffer B, and elution of aggregates at 100% Buffer B. The corresponding specific conductivities for these percentages of Buffer B were 5.5 mS/cm, 6.4 mS/cm, and 9.2 mS/cm for elution of empty particles, elution of full particles, and elution of aggregates, respectively.

The same approach was then taken with AAV8 as shown in Figure 8, with percentages as follows: wash at 0% Buffer B, elution of empty particles at 41.7% Buffer B, elution of full particles at 70% Buffer B, and elution of aggregates at 100% Buffer B.

EXAMPLE 6

Confirmation of empty and full AAV separation Although UV traces at 260/280 nm are indicative of empty/full separation, these results alone cannot be taken as proof positive without further investigation. To further characterize the separation, the anti-capsid B 1 antibody was employed in a western blot using a known quantity of AAV as the standard. The resulting blots show viral capsids in correlation to the peaks seen in the chromatogram (Figure 9). These results were compared to the same quantity of viral genome as measured by qPCR, and superimposed against the chromatogram. Results are shown in Figures 10A, 10B, and IOC.

EXAMPLE 7

Separation of AAV5, AAV6, and AAV8 Capsids

Recombinant AAV (rAAV) Production

Recombinant AAV particles were produced using the triple transfection method. 15- 24 hrs prior to transfection, 293 clones (SS10 Cells) were split to 70 % confluency in 15cm plates. Three plasmids (pHelper, pRC6, and pDISC-GFP) were combined at a molar ratio of 4.4: 3.3: 2.2. The plasmids were combined and mixed with Opti-MEM. PEI Max, at a ratio of 5:1 (w:w of DNA), was mixed with Opti-MEM. Both components were combined and vortexed, then allowed to incubate at room temperature for 15-30 minutes. The transfection cocktail, at 10 % of the culture volume, was then added to 15cm tissue culture treated dishes and allowed to incubate for 5 days at 37°C. rAAV Affininty Purification

Transfected cells were harvested and freeze thawed three times to release the viral particles. Cells were spun down at 7K RPM in a Sorvall RC-5B for 60 minutes, then filtered with a 0.2uM 1L filter device in preparation for chromatography. All subsequent chromatography experiments were performed on the AKTA Avant 150 using Unicorn 7.0. The crude cell lysate was captured on a GE HiScale 16 column packed with POROS™ CaptureSelect™ AAVX resin from Thermofisher and eluted in a low pH buffer. The peak fractions were identified using quantitative PCR and pooled. The pooled fractions were diluted 20 fold in 20mM Bis-Tris propane to raise the pH to 8.5. Although this resin specifically isolates AAV particles, it is not able to distinguish between empty and full particles. Thus, the elution peak will be mainly composed of empty and full particles of the specific serotype of AAV corresponding to the RC plasmid. The AAVX resin is specific for many different AAV serotypes, thus this method of capture can be employed irrespective of serotype. r AAV Anion Exchange purification

It has been shown that a chromatogram can visually depict the separation of empty and full rAAV particles. The absorption in milli-Absorbtion Units (mAU) at 260 nm typically depicts nucleic acid whereas absorption at 280 nm typically depicts protein. If the main component of the load material is AAV then if separation is indeed occurring, there will be a transition in the 260/280 trace. Where the population is mainly empty, the 280 nm trace will absorb higher than the 260nm. The converse is true of full particles; the 260 nm trace will absorb higher than 280nm. Because anion exchange chromatography is being used and the pi of an empty particle (about 6.3) is slightly higher than a full particle (about 5.9), the resulting separation will always occur in this protocol with empty particles eluting before the full particles.

Although many different modalities and resins have been shown to function for this application, the BIA separations monolith was chosen. The CIMmultus™ QA resin is manufactured prepacked in several different sizes, but for the purposes of these experiments, the 1ml monolith was chosen.

For initial screening, chromatographic runs were composed of equilibration, load, wash, elution in a linear gradient, and a strip step. The load, wash and strip were collected as pools whereas the elution was collected in 1ml fractions in a 96 deep well plate for ease of sampling for qPCR. Once positive fractions were identified, the elution profile was changed to an isocratic gradient allowing for efficient separation.

Quantitative PCR

To assess the quantity of viral DNA in each fraction, 5 pi of each fraction was sampled and digested with DNase to remove any non-viral or unpackaged DNA. The DNase was inactivated by incubation at 75°C and addition of EDTA. The samples were then further treated with Proteinase K to release packaged DNA and incubated at 55°C for 2 hours. The Proteinase K was inactivated by incubation at 95°C for 15 minutes. The samples were then diluted 10 and 100 fold and loaded onto a PCR plate and ran against a known quantity of plasmid DNA that was used to generate a standard curve. The PCR probes were directed toward a known sequence within the pDISC-GFP gene, and quantities were based on Ct values generated by the plasmid standard curve. Western blotting

To assess the quantity of capsid protein, 100 pi of each fraction was placed in a Dot- Blot apparatus and blotted onto Hybond ECL nitrocellulose membranes. The blots were then blocked with 10% milk, washed and probed with B1 monoclonal antibody (LSBio cat. no. C193393) which is specific for AAV VP1+VP2+VP3 capsid proteins. The blots were then washed and counter probed with an anti-mouse HRP monoclonal antibody, treated with ECL Prime and visualized using the UVP ChemiDoc-It2. In addition to samples, a known quantity of vims was serial diluted and blotted to establish a standard curve.

EXAMPLE 8

Separation of AAV9 Capsids

Following successful separation of AAV5, AAV6, and AAV8 capsids as described in Example 7, this method was applied for separation of AAV9 capsids. A single- stranded genome AAV9 was generated utilizing the triple transfection approach and purified using affinity chromatography. The affinity peak was then diluted and loaded onto the CIMmultus™ QA column and eluted using a linear gradient. The linear gradient was run using Buffer A (10 mM Bis-Tris propane 2 mM Magnesium chloride) and Buffer B (10 mM Bis-Tris propane 40 mM Magnesium chloride). The gradient was conducted from 0 to 100% of Buffer B over 65 CV.

A peak with an A260/280 ratio indicative of an empty AAV sample resulted at a conductivity of 2.2 mS/cm. A peak with an A260/280 ratio indicative of a full AAV sample resulted at a conductivity of 7.2 mS/cm.

Claims

1. A method for separating empty and full recombinant adeno-associated vims (rAAV) particles, said method comprising:

(a) loading a viral sample comprising empty rAAV particles and full rAAV particles on an anion exchange chromatography medium; and

(b) separately eluting said empty rAAV particles and said full rAAV particles using an isocratic elution gradient of magnesium chloride, wherein said isocratic elution gradient does not comprise one or more additional salts having a total concentration which affects separation of said empty rAAV particles and said full rAAV particles.

2. The method of claim 1, wherein said rAAV comprises serotype 5, 6, 8, or 9.

3. The method of claim 1 or claim 2, wherein said full rAAV comprises a gene of interest.

4. The method of claim 3, wherein said gene of interest encodes an engineered nuclease.

5. The method of claim 3 or claim 4, wherein said engineered nuclease is an engineered meganuclease, a TALEN, a compact TALEN, a zinc finger nuclease, a megaTAL, or a CRISPR system nuclease.

6. The method of claim 3, wherein said gene of interest encodes a chimeric antigen receptor or an exogenous T cell receptor.

7. The method of any one of claims 1-6, wherein said anion exchange chromatography medium comprises a monolithic column.

8. The method of claim 7, wherein said monolithic column comprises methacrylate.

9. The method of claim 8, wherein said methacrylate comprises poly(glycidyl methacrylate-co-ethylene dimethacrylate) .

10. The method of any one of claims 1-9, wherein said anion exchange chromatography medium comprises a quaternary amine.

11. The method of claim 10, wherein said anion exchange chromatography medium comprises the quaternary amine of CIMmultus™ QA or CIM^® QA.

12. The method of claim 11, wherein said anion exchange chromatography medium comprises CIMmultus™ QA or CIM^® QA.

13. The method of any one of claims 1-12, wherein said loading is performed in the presence of a first isocratic buffer comprising Bis-Tris propane.

14. The method of claim 13, wherein said isocratic elution gradient comprises increasing the percentage of a second isocratic buffer to said first isocratic buffer, wherein said second isocratic buffer comprises Bis-Tris propane and magnesium chloride, and wherein said second isocratic buffer does not comprise one or more additional salts having a total concentration which affects separation of said empty rAAV particles and said full rAAV particles.

15. The method of claim 14, wherein the concentration of magnesium chloride in said second isocratic buffer is between 1 mM and 100 mM.

16. The method of claim 15, wherein the concentration of magnesium chloride in said second isocratic buffer is between 20 mM and 60 mM.

17. The method of claim 16, wherein the concentration of magnesium chloride in said second isocratic buffer is about 40 mM.

18. The method of any one of claims 14-17, wherein the concentration of said Bis- Tris propane in said first isocratic buffer and said second isocratic buffer is between 5 mM and 30 mM.

19. The method of claim 18, wherein the concentration of said Bis-Tris propane in said first isocratic buffer and said second isocratic buffer is about 20 mM.

20. The method of any one of claims 14-19, wherein the pH of said Bis-Tris propane in said first isocratic buffer and said second isocratic buffer is between 8 and 9.

21. The method of claim 20, wherein the pH of said Bis-Tris propane is about 8.5.

22. The method of claim 14, wherein said first isocratic buffer comprises Bis-Tris propane at a concentration of about 20 mM and at a pH of about 8.5, and wherein said second isocratic buffer comprises Bis-Tris propane at a concentration of about 20mM and at a pH of about 8.5 and magnesium chloride at a concentration of about 40 mM.

23. The method of any one of claims 1-22, wherein said rAAV comprises serotype 6 and elution of empty rAAV particles occurs at a conductivity of about 6.2 mS/cm and elution of full rAAV particles occurs at a conductivity of about 7.8 mS/cm.

24. The method of claim 23, wherein elution of empty rAAV particles occurs at about 58.5% second isocratic buffer and elution of full rAAV particles occurs at about 80% second isocratic buffer.

25. The method of any one of claims 1-22, wherein said rAAV comprises serotype 5 and elution of empty rAAV particles occurs at a conductivity of about 5.5 mS/cm and elution of full rAAV particles occurs at a conductivity of about 6.4 mS/cm.

26. The method of claim 25, wherein elution of empty rAAV particles occurs at about 50.0% second isocratic buffer and elution of full rAAV particles occurs at about 61% second isocratic buffer.

27. The method of any one of claims 1-22, wherein said rAAV comprises serotype 8 and elution of empty rAAV particles occurs at a conductivity of about 4.9 mS/cm and elution of full rAAV particles occurs at a conductivity of about 7.0 mS/cm.

28. The method of claim 27, wherein elution of empty rAAV particles occurs at about 41.7% second isocratic buffer and elution of full rAAV particles occurs at about 70% second isocratic buffer.

29. The method of any one of claims 1-28, wherein said method further comprises loading said viral sample onto said anion exchange chromatography medium and washing said anion exchange chromatography medium to remove unbound fraction of said viral sample prior to elution.

30. The method of claim 29, wherein the pH of said viral sample is increased to between 8 and 9 with Bis-Tris propane prior to loading said viral sample onto said anion exchange chromatography medium.

31. The method of claim 30, wherein the pH of said viral sample is increased to about 8.5 with Bis-Tris propane prior to loading said viral sample onto said anion exchange chromatography medium.

32. The method of any one of claims 1-31, wherein said method further comprises performing a previous anion exchange chromatography prior to the anion exchange chromatography using said isocratic gradient, wherein said previous anion exchange chromatography comprises:

(a) loading a fraction of said viral sample onto said anion exchange chromatography medium; and

(b) eluting said viral sample using a linear elution gradient of a buffer comprising magnesium chloride to identify conductivity points for said isocratic elution gradient; wherein the first step of said isocratic elution gradient comprises the conductivity at which absorbance at 280 nm is greater than absorbance at 260 nm and absorbance at 280 nm is at its peak ± up to 0.5 mS/cm; and wherein the second step of said isocratic elution gradient comprises the conductivity at which absorbance at 260 nm is greater than absorbance at 280 nm and absorbance at 260 nm is at its peak ± up to 0.5 mS/cm; and wherein said linear elution gradient does not comprise one or more additional salts having a total concentration which affects separation of said empty rAAV particles and said full rAAV particles.

33. The method of claim 32, wherein said linear elution gradient comprises increasing the percentage of a second linear buffer to a first linear buffer, wherein said second linear buffer comprises Bis-Tris propane and magnesium chloride, and wherein said second linear buffer does not comprise one or more additional salts having a total concentration which affects separation of said empty rAAV particles and said full rAAV particles.

34. The method of claim 33, wherein the concentration of magnesium chloride in said second linear buffer is between 1 mM and 100 mM.

35. The method of claim 34, wherein the concentration of magnesium chloride in said second linear buffer is between 20 mM and 60 mM.

36. The method of claim 35, wherein the concentration of magnesium chloride in said second linear buffer is about 40mM.

37. The method of any one of claims 33-36, wherein the concentration of said Bis- Tris propane in said first linear buffer and said second linear buffer is between 5 mM and

30 mM.

38. The method of claim 37, wherein the concentration of said Bis-Tris propane in said first linear buffer and said second linear buffer is about 20 mM.

39. The method of any one of claims 33-38, wherein the pH of said Bis-Tris propane in said first linear buffer and said second linear buffer is between 8 and 9.

40. The method of claim 39, wherein the pH of said Bis-Tris propane is about 8.5.

41. The method of any one of claims 1-40, wherein said viral sample comprises host cell lysate.

42. The method of any one of claims 1-40, wherein said viral sample comprises an enriched eluate from an affinity chromatography of host cell lysate, wherein said enriched eluate is enriched for rAAV particles.

43. The method of claim 42, wherein said method further comprises performing said affinity chromatography of host cell lysate using an affinity chromatography medium specific for AAV, eluting with a low pH buffer, and collecting said enriched eluate from said affinity chromatography medium.

44. The method of claim 42 or claim 43, wherein presence of full rAAV particles in said enriched eluate from affinity chromatography is confirmed by performing quantitative PCR with primers specific for viral genomic DNA or said gene of interest.

45. The method of any one of claims 1-44, wherein said method further comprises collecting an enriched eluate from said anion exchange chromatography medium comprising full rAAV particles.

46. The method of claim 45, wherein presence of full rAAV particles in said enriched eluate from said anion exchange chromatography is confirmed by performing quantitative PCR with primers specific for viral genomic DNA or said gene of interest.

47. The method of any one of claims 1-45, wherein said rAAV comprises an rAAV5 particle.

48. The method of any one of claims 1-45, wherein said rAAV comprises an rAAV6 particle.

49. The method of any one of claims 1-45, wherein said rAAV comprises an rAAV8 particle.

50. The method of any one of claims 1-45, wherein said rAAV comprises an rAAV9 particle.

51. A viral composition comprising full rAAV particles produced by the method of any one of claims 1-50.

52. The viral composition of claim 51, wherein said viral composition comprises a higher ratio of full rAAV particles to empty rAAV particles when compared to a viral composition produced using another method of full rAAV purification.

53. A pharmaceutical composition comprising said viral composition of claim 51 or 52 and a pharmaceutically acceptable carrier.

54. A method for delivering a gene of interest, wherein said method comprises contacting a cell with the viral composition of claim 51 or 52 or the pharmaceutical composition of claim 53, and wherein said rAAV particle comprises said gene of interest.

55. The method of claim 54, wherein said gene of interest encodes an engineered nuclease.

56. The method of claim 55, wherein said engineered nuclease is an engineered meganuclease, a TALEN, a compact TALEN, a zinc finger nuclease, a megaTAL, or a CRISPR system nuclease.

57. The method of claim 54, wherein said gene of interest encodes a chimeric antigen receptor or an exogenous T cell receptor.

58. A method for treating a disease, wherein said method comprises administering to a subject in need thereof an effective amount of said pharmaceutical composition of claim 53, and wherein expression of said gene of interest treats said disease.

59. The viral composition of claim 51 or 52 for use as a medicament.

60. The viral composition for use according to claim 59, wherein said medicament is useful for treating a disease in a subject in need thereof, wherein expression of said gene of interest treats said disease.