WO2023028035A1 - Particules d'aav recombinants enveloppées de lipides pour une utilisation en thérapie génique - Google Patents

Particules d'aav recombinants enveloppées de lipides pour une utilisation en thérapie génique Download PDF

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WO2023028035A1
WO2023028035A1 PCT/US2022/041176 US2022041176W WO2023028035A1 WO 2023028035 A1 WO2023028035 A1 WO 2023028035A1 US 2022041176 W US2022041176 W US 2022041176W WO 2023028035 A1 WO2023028035 A1 WO 2023028035A1
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raav particles
aav
lipid enveloped
composition
raav
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PCT/US2022/041176
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Robert Mckenna
Mario MIETZSCH
Joshua Hull
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University Of Florida Research Foundation, Incorporated
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
    • A61K9/5068—Cell membranes or bacterial membranes enclosing drugs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • viruses exit host cells by forming a lipid membrane around the capsid, which membrane is typically derived from portions of the host cell membrane (phospholipids and proteins), but also may include some viral glycoproteins. These lipid membranes allow the enveloped virus to evade natural immune responses and amplify infectivity.
  • non-enveloped viruses such as the adeno-associated virus (AAV) typically exit host cells by cell lysis, and lipid membranes are not part of the released virions.
  • AAV adeno-associated virus
  • AAVs are non-enveloped viruses
  • the present inventors have surprisingly discovered rAAV particles which are embedded within a lipid envelope.
  • said lipid envelopes comprised single rAAV particles, unlike previously described exosomes comprising multiple rAAV particles.
  • the newly -discovered lipid enveloped rAAV particles may have increased multiplicity of infection (MOI) and enhanced immune evasion properties, relative to non-lipid enveloped rAAV particles, and therefore may be useful in the treatment of disease.
  • MOI multiplicity of infection
  • the present disclosure relates to such lipid enveloped rAAV particles, compositions comprising the same, and methods of treating a disease comprising the administration of such lipid enveloped rA AV particles and compositions. Methods of preparing compositions comprising a population of lipid enveloped rAAV particles are also disclosed.
  • aspects of the disclosure relate to a composition comprising a plurality of rAAV particles, wherein at least about 20% of the rAAV particles are lipid enveloped rAAV particles. In some embodiments, at least about 25%, at least about 30%, or at least about 35% of the rAAV particles in the composition comprising a plurality of rAAV particles are lipid enveloped rAAV particles. In some embodiments, at least about 10 11 , at least about 10 12 , at least about 10 13 , at least about 10 14 , or at least about 10 15 of the rAAV particles in the composition comprising a plurality of rAAV particles are lipid enveloped rAAV particles.
  • a composition comprising lipid enveloped rAAV particles further comprises a pharmaceutically -acceptable earner or excipient. In some embodiments, the composition is sterilized.
  • the composition (a) has enhanced immune evasion properties: (b) is less immunogenic; (c) has greater infectivity; and/or (d) has a greater multiplicity of infection (MOI), relative to a heterogenous composition of rAAV particles comprising non-lipid enveloped rAAV particles and/or vesicles or exosomes each comprising multiple rAAV particles.
  • MOI multiplicity of infection
  • the method comprises: (a) producing a. heterogenous population of rAAV particles comprising non-lipid enveloped and lipid enveloped rAAV particles in a host producer cell comprised in a .medium; (b) harvesting the medium supernatant, wherein the medium supernatant comprises lipid enveloped rAAV particles; and (c) isolating the lipid enveloped rAAV particles.
  • the host producer cell is a mammalian or insect cell.
  • the mammalian cell is a HEK293 cell.
  • the insect cell is a mammalian or insect cell.
  • the method of preparing a population of lipid enveloped rAAV particles further comprises a step of overexpressing membrane associated accessory protein (MAAP) and/or CD9 in the host producer cell.
  • MAAP membrane associated accessory protein
  • both MAAP and CD9 are overexpressed. In some embodiments, either MAAP or CD9 is overexpressed.
  • the medium supernatant does not comprise non-lipid enveloped rAAV particles.
  • step (b) does not comprise harvesting the host producer cells.
  • the method further comprises a step of adding fresh medium to the host producer cells, and the medium supernatant is re -harvested.
  • the step of adding fresh medium and re-harvesting is repeated 1 time, 2 times, 3 times, or 4 times.
  • isolating the lipid enveloped rAAV particles comprises precipitating the lipid enveloped rAAV particles and/or purifying the precipitate.
  • the lipid enveloped rAAV particles are precipitated using polyethylene glycol 8000 (PEG8000).
  • PEG8000 is in a concentration of at least about 5%, at least about 10%, or at least about 15% weight per volume (w/v).
  • purifying the precipitate comprises performing iodixanol gradient ultracentrifugation or sucrose- gradient ultracentri fugation .
  • At least about 20%', at least about 25%, at least about 30%, or at least about 35% of the isolated rAAV particles in the composition comprising a plurality of rAAV particles are lipid enveloped rAAV particles.
  • at least about 10 11 , at least about 10 !2 , at least about 10 ij , at least about 10 14 , or at least about 10 15 of the isolated rAAV particles in the composition comprising a plurality of rAAV particles are lipid enveloped rAAV particles.
  • the AAV is AAV serotype 2 (AAV2).
  • the AAV is AAV serotype 1 (AAV1), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (A.AV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), AAV serotype 12 (AAV12), AAV serotype 13 (AAV13), AAV rhesus isolate 8 (AAVrh.8), AAV rhesus isolate 10 (AAVrh.10), AAV rhesus isolate 39 (AAVrh.39), or AAV rhesus isolate 74 (AAVrh.74 ).
  • the heterogenous population of rAAV particles further comprises vesicles or exosomes each comprising multiple rAAV particles.
  • the harvested medium supernatant does not comprise vesicles or exosomes each comprising multiple rAAV particles.
  • the harvested medium supernatant does not comprise (i) non-lipid enveloped rAAV particles nor (ii) vesicles or exosomes each comprising multiple rAAV particles.
  • aspects of the di sclosure relate to a method of treating a disease or disorder comprising administering a composition comprising lipid enveloped rAA V particles as described herein, wherein the lipid enveloped rAAV particles in the composition comprise a nucleic acid encoding a therapeutic molecule.
  • the disease or disorder is a cancer, diabetes, autoimmune disease, kidney disease, cardiovascular disease, pancreatic disease, intestinal disease, liver disease, neurological disease, neuromuscular disorder, neuromotor deficit, neuroskeletal impairment, neurological disability, neurosensory dysfunction, stroke, al-antitrypsin (AAT) deficiency.
  • the subject is a human subject.
  • a lipid enveloped rAAV particle or composition of the disclosure has enhanced immune evasion properties or is less immunogenic than a non-lipid enveloped rAAV particle or heterogenous composition of rAAV particles comprising non-lipid enveloped rAAV particles. In some embodiments, a lipid enveloped rAAV particle or composition of the disclosure has greater infectivity than a non-lipid enveloped rAAV particle or heterogenous composition of rAAV particles comprising non-lipid enveloped rAAV particles.
  • a lipid enveloped rAAV particle or composition of the disclosure has a greater multiplicity of infection (MOI) than a non-lipid enveloped rAAV particle or heterogenous composition of rAAV particles comprising non-lipid enveloped rAAV particles.
  • MOI multiplicity of infection
  • composition comprising lipid enveloped rAAV particles as described herein is administered to the subject one time or multiple times.
  • Figures 1A-1B show enveloped particle identification.
  • Figure 1A shows negative stain electron microscopy (EM) of particles, revealing the presence of both lipid enveloped (white arrows) and non-enveloped (black arrows) virus-like particles (VLPs). Particles are ordered in sub-panels (i)-(iii) by sphericity. Envelopes are stain-impermeable, leaving their interior contents obscured.
  • Figure IB shows cryo-electron microscopy (cryo-EM) of capsids, which again demonstrated the presence of lipid enveloped AAV2 VLPs.
  • Exemplary spheroid particles (i)-(iv) were selected for single particle reconstruction, with broken (v) and empty (vi) envelopes also observed (but not selected).
  • White arrows (in (v) and (vi)) indicate the ends of broken envelopes.
  • Scale bar depicts 200 ⁇ .
  • Figures 3A-3F show structural characterization of the lipid enveloped AAV capsid.
  • Figure 3A show's the final map of the lipid enveloped AAV particle at 3.14 ⁇ resolution, contoured to 3o, overlaid onto the lipid envelope map at 10 ⁇ resolution, contoured to l ⁇ .
  • the capsid is shaded using a radial distance scale.
  • the generalized locations of the envelope and AAV capsid are indicated by arrow's.
  • the 2-fold (oval), 3-fold (triangle), and 5-fold (pentagon) are indicated to indicate the asymmetric unit of the capsid.
  • the open trapezoid in the upper left quadrant highlights the region of the structure shown in Figure 3E.
  • Figure 3B shows the non- enveloped AAV capsid final map at 2.43 ⁇ resolution, contoured to 3 ⁇ , using the same radial distance scale.
  • Figures 3C and 3D show the P-strand G (PG) for the enveloped ( Figure 3C) and non-enveloped AAV (Figure 3D) contoured to 2 ⁇ to demonstrate map quality of the structures, respectively.
  • Figure 3E shows a zoomed-in view of the trapezoid shown in Figure 3.A, to demonstrate the fit of the structure.
  • the low-resolution map is shown contoured to lo with a diacylglycerol and phospholipid (POPC) lipid membrane docked (CC 0.52 at 10 ⁇ resolution) to demonstrate that a lipid bilayer can readily fit into the halo envelope density. Also shown is the docked enveloped AAV capsid structure (CC 0.92 at 10 ⁇ resolution).
  • the AAV2 VP3 variable regions (VRs) IV, V, and VIII are indicated by arrows.
  • Figure 3F shows a close-up of the open boxed region of Figure 3E, with side chains shown. Distances are given of the closest approach of VRs to the modeled lipid envelope as docked. Examples of the following amino acid side chains are indicated by arrows: oxygen, phosphate, nitrogen.
  • the present disclosure relates to lipid enveloped rAAV particles, compositions comprising the same, and methods of treating a disease comprising the administration of such lipid enveloped rAAV particles and compositions. Methods of preparing compositions comprising lipid enveloped rAAV particles are also disclosed.
  • Lipid enveloped rAAV particles Lipid enveloped rAAV particles
  • lipid enveloped rAAV particles refers to a single rAAV particle surrounded by a lipid membrane. It is specifically contemplated that the lipid enveloped rAAV particles of the disclosure do not comprise multiple rAAV particles per lipid envelope.
  • the lipid enveloped rAAV particles of the disclosure are approximately 40 nm in diameter.
  • a lipid enveloped rAAV particle of the disclosure is approximately 35 nm, approximately 36 nm, approximately 37 nm, approximately 38 nm, approximately 39 nm, approximately 40 nm, approximately 41 nm, approximately 42 nm, approximately 43 nm, approximately 44 nm, approximately 45 nm, approximately 46 nm, approximately 47 nm, approximately 48 nm, approximately 49 nm, approximately 50 nm, approximately 51 nm, approximately 52 nm, approximately 53 nm, approximately 54 nm, approximately 55 nm, approximately 56 nm, approximately 57 nm, approximately 58 nm, approximately 59 nm, approximately 60 nm, approximately 61 nm, approximately 62 nm, approximately 63 nm, approximately 64 nm, approximately 65 nm, approximately 66 nm, approximately 67 nm, approximately 68 nm, approximately 69 nm, approximately 70 nm, approximately 71 nm, approximately 72
  • a lipid enveloped rAAV particle of the disclosure is about 35 nm to about 37 nm in diameter, about 36 nm to about 38 nm in diameter, about 37 nm to about 39 nm in diameter, about 38 nm to about 40 nm in diameter, about 39 nm to about 41 nm in diameter, about 40 nm to about 42 nm in diameter, about 41 nm to about 43 nm in diameter, about 42 nm to about 44 nm in diameter, about 43 nm to about 45 nm in diameter, about 44 nm to about 46 nm in diameter, about 45 nm to about 47 nm in diameter, about 46 nm to about 48 nm in diameter, about 47 nm to about 49 nm in diameter, about 48 nm to about 50 nm in diameter, about 49 nm to about 51 nm in diameter, about 50 nm to about 52 nm in diameter, about 51 nm to about 53 nm
  • a lipid enveloped rAAV particle of the disclosure is about 30 nm to about 50 nm in diameter. In some embodiments, a lipid enveloped rAAV particle of the disclosure is less than 250 nm in diameter, less than 240 nm in diameter, less than 230 nm in diameter, less than 220 nm in diameter, less than 210 nm in diameter, less than 200 nm in diameter, less than 190 nm in diameter, less than 180 nm in diameter, less than 170 nm in diameter, less than 160 nm in diameter, less than 150 nm in diameter, less than 140 nm in diameter, less than 130 nm in diameter, less than 120 nm in diameter, less than 110 nm in diameter, or less than 100 nm in diameter.
  • a lipid enveloped rAAV particle of the disclosure is less than 200 nm in diameter. In some embodiments, a lipid enveloped rAAV particle of the disclosure is less than 150 nm in diameter. In some embodiments, a lipid enveloped rAAV particle of the disclosure is less than 100 nm in diameter. In some embodiments, a lipid enveloped AAV particle comprises an empty capsid (e.g., a capsid without a cargo). In some embodiments, a lipid enveloped rAAV particle comprises a capsid encapsidating a nucleic acid (e.g., a nucleic acid encoding a therapeutic molecule of interest), as described elsewhere herein.
  • a nucleic acid e.g., a nucleic acid encoding a therapeutic molecule of interest
  • a lipid enveloped rAAV particle disclosed herein comprises a capsid protein comprising one or more mutations, e.g., one or more amino acid substitutions, insertions, and/or deletions.
  • a lipid enveloped rAAV particle disclosed herein comprises one single-stranded DNA.
  • a lipid enveloped rAAV particle disclosed herein comprises two complementary DNA strands, forming a self-complementary AAV (scAAV). in some embodiments, a lipid enveloped rAAV particle disclosed herein is replicative.
  • a replicative lipid enveloped rAAV particle is capable of replicating within a host cell (e.g., a host cell within a subject or a host cell in culture).
  • a lipid enveloped rAAV particle disclosed herein is non-replicating.
  • a non-replicating lipid enveloped rAAV particle is not capable of replicating within a host cell (e.g., a host cell within a subject or a host cell in culture), but can infect the host and incorporate genetic components into the host’s genome for expression.
  • a lipid enveloped rAAV particle disclosed herein is capable of infecting a host cell.
  • a lipid enveloped rAAV particle disclosed herein is capable of facilitating stable integration of genetic components into the genome of a host cell. In some embodiments, a lipid enveloped rAAV particle disclosed herein is not capable of facilitating integration of genetic components into the genome of a host cell.
  • a lipid enveloped rAAV particle disclosed herein may be of any AAV serotype (e.g., AAV serotype 1, 2, 3. 4, 5, 6, 7, 8, 9, 10, 11 , 12, or 13), including any derivative (including non- naturally occurring variants of a serotype) or pseudotype.
  • Non-limiting examples of derivatives and pseudotypes include AAV2-AAV3 hybrid, AAVrh.10, AAVhu.14, AAV3a/3b, AAVrh32.33, AAV-HSC15, AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15/17, AAVM41, AAV9.45, AAV2.5T, AAV-HAE1/2, AAV clone 32/83, AAVShH10, AAV2.15, AAV2.4, AAVM41, and AAVr3.45.
  • AAV serotypes and derivatives/pseudotypes are known in the art (see, e.g., Mol. Ther. 2012 Apr; 20(4):699-708. doi: 10.1038/mt.2011.287. Epub 2012 Jan 24.
  • the AAV vector toolkit poised at the clinical crossroads. Asokan A, Schaffer DV, Samuiski RJ.).
  • the AAV particle is a pseudotyped AAV particle, which comprises a nucleic acid vector comprising ITRs from one serotype (e.g., AAV2 or AAV3) and a capsid comprised of capsid proteins derived from another serotype (i.e., a serotype other than AAV2 or AAV3, respectively).
  • a pseudotyped AAV particle which comprises a nucleic acid vector comprising ITRs from one serotype (e.g., AAV2 or AAV3) and a capsid comprised of capsid proteins derived from another serotype (i.e., a serotype other than AAV2 or AAV3, respectively).
  • the AAV is AAV serotype 2 (AAV2).
  • the AAV is AAV serotype 1 (AAV1 ), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAV11 ), AAV serotype 12 (AAV12), AAV serotype 13 (AAV13), AAV rhesus isolate 8 (AAVrh.8), AAV rhesus isolate 10 (AAVrh.10), AAV rhesus isolate 39 (AAVrh.39), or AAV rhesus isolate 74 (AAVrh.74).
  • rAAV particles may comprise a nucleic acid vector which may comprise at a minimum (a) one or more heterologous nucleic acid regions comprising a sequence encoding a therapeutic molecule of interest (described elsewhere herein) and (b) one or more regions comprising inverted terminal repeat (ITR) sequences (e.g., wild-type ITR sequences or engineered ITR sequences) flanking the one or more heterologous nucleic acid regions.
  • the nucleic acid vector is between 4 kb and 5 kb in size (e.g., 4.2 to 4.7 kb in size).
  • This nucleic acid vector may be encapsidated by a viral capsid, such as an AAV2 capsid.
  • the nucleic acid vector is circular.
  • the nucleic acid vector is single-stranded.
  • the nucleic acid vector is double-stranded.
  • a double- stranded nucleic acid vector may be, for example, a self-complimentary vector that contains a region of the nucleic acid vector that is complementary' to another region of the nucleic acid vector, initiating the formation of the double-strandedness of the nucleic acid vector.
  • a lipid enveloped rAAV particle of the disclosure comprises a viral capsid and a nucleic acid vector, which is encapsidated by the viral capsid.
  • the nucleic acid vector comprises (1) one or more heterologous nucleic acid regions comprising a sequence encoding a therapeutic molecule, (2) one or more nucleic acid regions comprising a sequence that facilitates expression of the heterologous nucleic acid region(s) (e.g., a promoter), and (3) one or more ITR sequences.
  • the nucleic acid vector comprises one or more heterologous nucleic acid regions comprising a sequence encoding a therapeutic molecule operably linked to a promoter, wherein the one or more heterologous nucleic acid regions are flanked on each side with an ITR sequence.
  • a nucleic acid vector comprised in a lipid enveloped rAAV particle further comprises one or more AAV ITRs. In some embodiments, a nucleic acid vector comprised in a lipid enveloped rAAV particle comprises two AAV ITRs. In some embodiments, the AAV ITR(s) are naturally-occurring AAV ITRs. In some embodiments, the AAV ITR(s) are synthetic AAV ITR(s).
  • the ITR sequences can be derived from any AAV serotype (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) or can be derived from more than one serotype. In some embodiments, the ITR sequences are derived from AAV2 or AAV6.
  • ITR sequences and plasmids containing ITR sequences are known in the art and commercially available (see, e.g., products and services available from Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, CA: and Addgene, Cambridge, MA; and Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein.
  • Kessler PD Podsakoff GM, Chen X, McQuiston SA, Colosi PC, Matelis LA, Kurtzman GJ, Byrne BJ. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24): 14082-7; and Curtis A. Machida. Methods in Molecular MedicineTM.
  • a lipid enveloped rAAV particle of the disclosure has enhanced immune evasion properties or is less immunogenic than a non-lipid enveloped rAAV particle and/or than vesicles or exosomes each comprising multiple rAAV particles. In some embodiments, a lipid enveloped rAAV particle of the disclosure has greater infectivity than a non-lipid enveloped rAAV particle and/or than vesicles or exosomes each comprising multiple rAAV particles.
  • a lipid enveloped rAAV particle of the disclosure has a greater multiplicity of infection (MOI) than a non-lipid enveloped rAAV particle and/or than vesicles or exosomes each comprising multiple rAAV particles.
  • a lipid enveloped rAAV particle of the disclosure has enhanced immune evasion properties or is less immunogenic than, has greater infectivity than, and/or has a greater multiplicity of infection (MOI) than a non-lipid enveloped rAAV particle and/or than vesicles or exosomes each comprising multiple rAAV particles.
  • the non-lipid enveloped rAAV particle of any of the foregoing embodiments is of the same serotype as the lipid enveloped rAAV particle.
  • the rAAV particles e.g., non-lipid enveloped rAAV particles, and/or rAAV particles comprised in vesicles or exosomes
  • the rAAV particles are of the same serotype and comprise the same cargo as the lipid enveloped rAAV particles.
  • Virus neutralization assays are known in the art, and are a specialized type of immunoassay that can detect certain antibodies which block or inhibit virus replication (e.g., an antibody which can neutralize virus infection).
  • a virus neutralization assay may be used in conjunction with a viral infectivity assay (e.g., a luciferase reporter assay, a plaque assay) to determine the infectivity of a viral particle in the presence of said neutralizing antibodies.
  • Viral infectivity assays are known in the art, and measure the ability of a virus to productively infect a cell.
  • the results of the virus neutralization and/or viral infectivity assay is visualized through the detection of a fluorescent or otherwise detectable marker, such as a luciferase or other fluorescent protein (e.g., green fluorescent protein, or any other fluorescent protein as described herein).
  • a fluorescent or otherwise detectable marker such as a luciferase or other fluorescent protein (e.g., green fluorescent protein, or any other fluorescent protein as described herein).
  • the transduction rate of a lipid enveloped rAAV particle of the disclosure, measured using a virus neutralization and/or viral infectivity assay as described herein is higher than that of a non-lipid enveloped rAAV particle in the presence of neutralizing antibodies.
  • the neutralization curve for a lipid enveloped rAAV particle of the disclosure is shifted significantly (e.g., by 1 log of antibody concentration, or more) relative to that of a non-lipid enveloped rAAV particle in the presence of neutralizing antibodies.
  • the non-lipid enveloped rAAV particle of any of the foregoing embodiments is of the same serotype as the lipid enveloped rAAV particle.
  • the non-lipid enveloped rAAV particles of any of the foregoing embodiments is of the same serotype and comprises the same cargo as the lipid enveloped rAAV particle.
  • compositions comprising jipid enveloped rAAV particles
  • compositions comprising a plurality of lipid enveloped rAAV particles. Such compositions may be useful in the delivery of therapeutic molecules to subjects in need thereof, as described elsewhere herein. In some embodiments, the compositions comprising a plurality of lipid enveloped rAAV particles are produced according to the methods described elsewhere herein.
  • At least about 20% of the rAAV particles in the composition comprising a plurality of rAAV particles are lipid enveloped rAAV particles. In some embodiments, 100% of the rAAV particles in the composition comprising a plurality of rAAV particles are lipid enveloped rAAV particles.
  • a composition comprising lipid enveloped rAAV particles may also comprise non-lipid enveloped rAAV particles and/or vesicles or exosomes comprising multiple rAAV particles, in addition to comprising lipid enveloped rAAV particles.
  • the rAAV particles of the composition comprising lipid- enveloped rAAV particles which do not comprise lipid enveloped rAAV particles are non-lipid enveloped rAAV particles.
  • the rAAV particles of the composition comprising lipid-enveloped rAAV particles which do not comprise lipid enveloped rAAV particles are exosomes or liposomes comprising multiple rAAV particles.
  • At least about 10 7 , at least about 10 8 , at least about 10 9 , at least about 10 10 , at least about 10 11 at least about 10 12 , at least about 10 13 , at least about 10 14 , or at least about 10 13 of the rAAV particles in the composition comprising a plurality of rAAV particles are lipid enveloped rAAV particles.
  • At least about 10 7 to at least about 10 9 , at least about 10 8 to at least about 10 10 , at least about 10 9 to at least about 10 11 , at least about 10 10 to at least about 10 12 , at least about 10 11 to at least about 10 13 , at least about 10 12 to at least about 10 14 , or at least about 10 13 to at least about 10 13 of the rAAV particles in the composition comprising a plurality of rAAV particles are lipid enveloped rAAV particles.
  • a composition of the disclosure (e.g., comprising lipid enveloped rAAV particles) has enhanced immune evasion properties or is less immunogenic than a heterogenous composition of rAAV particles comprising non-lipid enveloped rAAV particles and/or vesicles or exosomes each comprising multiple rAAV particles.
  • a composition of the disclosure has greater infectivity than a heterogenous composition of rAAV particles comprising non-lipid enveloped rAAV particles and/or vesicles or exosomes each comprising multiple rAAV particles.
  • a composition of the disclosure has a greater multiplicity of infection (MOI) than a heterogenous composition of rAAV particles comprising non-lipid enveloped rAAV particles and/or vesicles or exosomes each comprising multiple rAAV particles.
  • a composition of the disclosure has enhanced immune evasion properties or is less immunogenic than, has greater infectivity than, and/or has a greater multiplicity of infection (MOI) than a heterogenous composition of rAAV particles comprising non-lipid enveloped rAAV particles and/or vesicles or exosomes each comprising multiple rAAV particles.
  • the rAAV particles e.g., non-lipid enveloped rAAV particles, and/or rAAV particles comprised in vesicles or exosomes
  • the rAAV particles are of the same serotype as the lipid enveloped rA AV particles.
  • the rAAV particles e.g., non-lipid enveloped rAAV particles, and/or rAAV particles comprised in vesicles or exosomes
  • the rAAV particles are of the same serotype and comprise the same cargo as the lipid enveloped rAAV particles.
  • a composition comprising lipid enveloped rAAV particles further comprises a pharmaceutically-acceptable carrier or excipient, as described elsewhere herein.
  • a composition comprising lipid enveloped rAAV particles is sterilized. Sterilization is a process of removing objectionable microorganisms and controlling microbial populations within a pharmaceutical composition. Methods of sterilization are well known in the art, and are described, for example, in Armenante, P.M. and Akiti, O. (2019). STERILIZATION PROCESSES IN THE PHARMACEUTICAL INDUSTRY. In Chemical Engineering in the Pharmaceutical Industry (eds M.T. Ende and D.J. Ende).
  • a composition comprising lipid enveloped rAAV particles is sterilized using sterile filtering of the AAV preparation. Methods of preparing a population of lipid enveloped rAAV partides
  • the method comprises: (a) producing a heterogenous population of rAAV particles comprising non-lipid enveloped and lipid enveloped rAAV particles in a host producer cell comprised in a medium; (b) harvesting the medium supernatant, wherein the medium supernatant comprises lipid enveloped rAAV particles; and (c) isolating the lipid enveloped rAAV particles.
  • the heterogenous population of rAAV particles further comprises vesicles or exosomes each comprising multiple rAAV particles.
  • the method comprises: (a) producing a heterogenous population of rAAV particles comprising (i) non-lipid enveloped rAAV particles and/or vesicles or exosomes each comprising multiple rAAV particles and (ii) lipid enveloped rAAV particles in a host producer cell comprised in a medium; (b) harvesting the medium supernatant, wherein the medium supernatant comprises lipid enveloped rAAV particles; and (c) isolating the lipid enveloped rAAV particles.
  • rAAV particles Methods of producing rAAV particles are known in the art and commercially available (see, e.g., Zolotukhin et al. Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28 (2002) 158—167; and U.S. Patent Publication Nos. US 2007/0015238 and US 2012/0322861, which are incorporated herein by reference; and plasmids and kits available from ATCC and Cell Biolabs, Inc.).
  • a plasmid containing the nucleic acid vector sequence may be combined with one or more helper plasmids, e.g., that contain a rep gene (e.g., encoding Rep78, Rep68, Rep52 and Rep40) and a cap gene (encoding VP1, VP2, and VP3, including a modified VP3 region as described herein), and transfected into a producer cell line such that the rAAV particle can be packaged and subsequently purified.
  • helper plasmids e.g., that contain a rep gene (e.g., encoding Rep78, Rep68, Rep52 and Rep40) and a cap gene (encoding VP1, VP2, and VP3, including a modified VP3 region as described herein)
  • the one or more helper plasmids includes a first helper plasmid comprising a rep gene and a cap gene and a second helper plasmid comprising a El a gene, a Elb gene, a E4 gene, a E2a gene, and a VA gene.
  • the rep gene is a rep gene derived from AAV2 and the cap gene is a cap gene derived from AAV2 and includes modifications to the gene in order to produce a modified capsid protein described herein.
  • Helper plasmids, and methods of making such plasmids are known in the art and commercially available (see, e.g., pDM, pDG, pDPlrs, pDP2rs, pDP3rs, pDP4rs, pDP5rs, pDP6rs, pDG(R484E/R585E), and pDP8.ape plasmids from PlasmidFactory, Bielefeld, Germany; other products and services available from Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, CA; and Addgene, Cambridge, MA; pxx6; Grimm et al.
  • helper plasmids are produced or obtained, which comprise rep and cap ORFs for the desired AAV serotype and the adenoviral VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters.
  • the cap ORF may also comprise one or more modifications to produce a modified capsid protein as described herein.
  • HEK293 cells available from ATCC® are transfected via CaPO4-mediated transfection, lipids or polymeric molecules such as Polyethylenimine (PEI) with the helper plasmid(s) and a plasmid containing a nucleic acid vector described herein.
  • PEI Polyethylenimine
  • HEK293 cells are then Incubated for at least 60 hours to allow for rAAV particle production.
  • Sf9-based producer stable cell lines are infected with a single recombinant baculo virus containing the nucleic acid vector.
  • HEK293 or BHK cell lines are infected with a HSV containing the nucleic acid vector and optionally one or more helper HSVs containing rep and cap ORFs as described herein and the adenoviral VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters.
  • the host producer cell is a mammalian or insect cell.
  • the mammalian cell is a HEK293 cell.
  • the insect cell is a Sf9 cell.
  • the host producer cell produces and comprises a heterogenous population of rAAV particles comprising non-lipid enveloped and lipid enveloped rAAV particles. In some embodiments, the host producer cell produces and comprises a heterogenous population of rAAV particles comprising (i) non-lipid enveloped rAAV particles and/or vesicles or exosomes each comprising multiple rAAV particles and (ii) lipid enveloped rAAV particles.
  • typical rAAV preparations include a step of lysing the host producer cell, or otherwise extracting the non-lipid enveloped rAAV particles and/or the rAAV particles multiply comprised within vesicles or exosomes from the cell, according to methods known in the art, while discarding the medium supernatant.
  • a host producer cell may secrete lipid enveloped rAAV particles into the rAAV production medium while retaining non-lipid enveloped rAAV particles and/or vesicles or exosomes each comprising multiple rAAV particles within the cell.
  • the lipid enveloped rAAV particles of the present disclosure are secreted from the cell, no step of lysis or extraction is necessary.
  • the present methods do not comprise a step of lysis or extraction (e.g., using detergents, freeze-thaw, or micro-fluidizing methods) so as to not disrupt or lyse the lipid envelopes surrounding the rAAV particles of interest.
  • the medium supernatant therefore comprises lipid enveloped rAAV particles.
  • the medium supernatant comprising lipid enveloped rAAV particles is harvested.
  • harvesting the medium supernatant does not comprise harvesting the host producer cells.
  • harvesting the medium supernatant does not comprise harvesting non-lipid enveloped rAAV particles.
  • harvesting the medium supernatant does not comprise harvesting vesicles or exosomes each comprising multiple rAAV particles.
  • fresh medium is added to the producer host cells (e.g., a second medium).
  • the medium supernatant is again harvested.
  • the addition of fresh medium and harvesting of the same is repeated 1 time, 2 times, 3 times, or 4 times.
  • certain proteins are overexpressed in the host producer cell in order to enrich or amplify production of lipid enveloped rAAV particles, for example by increasing the quantity of lipid enveloped rAAV particles released from the host producer cell.
  • the method of preparing a population of lipid enveloped rAAV particles further comprises a step of overexpressing membrane associated accessory protein (MAAP) and/or CD9 in the host producer cell.
  • MAAP membrane associated accessory protein
  • MAAP is overexpressed in the host producer cell.
  • MAAP is a unique, virally-encoded protein with an amphipathic, cationic membrane anchoring domain (see Ogden, et al. (2019) Comprehensive AAV capsid fitness landscape reveals a viral gene and enables machine -guided design, Science 366(6469): 1139-43).
  • MAAP is coded within an alternate reading frame of VP1 and has been shown to accumulate at the host cell membrane, and is believed to play a role in the natural life cycle of AAV — limiting AAV production through competitive exclusion — and is localized to the plasma membrane of infected cells.
  • the overexpression of MAAP in the host producer cell enriches the production of lipid enveloped rAAV particles. In some embodiments, the overexpression of MAAP in the host producer cell enhances the secretion of lipid enveloped rAAV particles from the host producer cell. In some embodiments, the overexpression of MAAP in the host producer cell enriches the production of and enhances the secretion of lipid enveloped rAAV particles from the host producer cell.
  • CD9 is overexpressed in the host producer cell.
  • CD9 is a protein that is a member of the transmembrane 4 superfamily also known as the tetraspanin family. It is a cell surface glycoprotein that consists of four transmembrane regions and has two extracellular loops that contain disulfide bonds which are conserved throughout the tetraspanin family. CD9 is involved in cell motility, adhesion, and fusion, and the loss of CD9 expression leads to tumor progression and metastasis in several types of cancer. CD9 has been shown to enhance exosome secretion in lenti viral contexts (see, e.g., Bdker, et al.
  • the overexpression of CD9 in the host producer cell enriches the production of lipid enveloped rAA V particles. In some embodiments, the overexpression of CD9 in the host producer cell enhances the secretion of lipid enveloped rAAV particles from the host producer cell. In some embodiments, the overexpression of CD9 in the host producer cell enriches the production of and enhances the secretion of lipid enveloped rAAV particles from the host producer cell. In some embodiments, both MAAP and CD9 are overexpressed in the host producer cell. In some embodiments, isolating the lipid enveloped rAAV particles comprises precipitating the lipid enveloped rAAV particles and/or purifying the precipitate.
  • the lipid enveloped rAAV particles are precipitated from the harvest medium supernatant.
  • Methods of precipitation are known in the art, and may comprise, for example, the use of polyethylene glycol (PEG).
  • the PEG is PEG8000 (available commercially at, for example, Sigma Aldrich, catalog # 1546605).
  • the PEG8000 is in a concentration of at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%', at least about 14%', or at least about 15% weight per volume (w/v).
  • the PEG8000 is in a concentration range of about 5% to about 7%, about 6% to about 8%', about 7% to about 9%, about 8% to about 10%, about 9% to about 11%, about 10% to about 12%, about 11% to about 13%, about 12% to about 14%, or about 13% to about 15% weight per volume (w/v). In some embodiments, the PEG8000 is in a concentration of 10% weight per volume (w/v).
  • the resulting precipitate is purified.
  • Methods of purification include, for example, iodixanol gradient ultracentrifugation or sucrose- gradient ultracentrifugation.
  • a sample containing a mixture of different size macromolecules is layered on the surface of a gradient whose density increases linearly from top to bottom.
  • the gradient may be an iodixanol gradient, or may be a sucrose gradient.
  • different size macromolecules sediment through the gradient at different rates. The rate of sedimentation depends, in addition to centrifugal force, on the size, shape, and density of the macromolecules, as well as on the density and viscosity of the gradient.
  • At least about 20% of the isolated rAAV particles are lipid enveloped rAAV particles. In some embodiments, 85% of the isolated rAAV particles are lipid enveloped rAAV particles. In some embodiments, 90% of the isolated rAAV particles are lipid enveloped rAAV particles. In some embodiments, 95% of the isolated rAAV particles are lipid enveloped rAAV particles. In some embodiments, 100% of the isolated rAAV particles are lipid enveloped rAAV particles.
  • At least about 10 7 , at least about 10 8 , at least about 10 9 , at least about 10 10 , at least about 10 31 , at least about 10 12 , at least about 10 13 , at least about 10 14 , or at least about 10 15 of the isolated rAAV particles are lipid enveloped rAAV particles.
  • At least about 10' to at least about 10 9 , at least about 10 8 to at least about 10 10 , at least about 10 9 to at least about 10 11 , at least about 10 10 to at least about 10 12 , at least about 10 11 to at least about 10 13 , at least about 10 12 to at least about 10 14 , or at least about 10 13 to at least about 10 15 of the isolated rAAV particles are lipid enveloped rAAV particles.
  • aspects of the invention include methods of treating a disease or disorder, the method comprising administering a lipid enveloped rAA V particle as described herein or a composition comprising a lipid enveloped rAAV particle as described herein to a subject in need thereof.
  • the lipid enveloped rAAV particles of the disclosure which are administered to a subject comprise a nucleic acid encoding a therapeutic molecule.
  • the lipid enveloped rAAV particles of the disclosure which are administered to a subject may also comprise a nucleic acid(s) encoding one or more regulatory elements.
  • a nucleic acid sequence encoding a regulatory element refers to a nucleotide sequence or structural component of a nucleic acid vector which is involved in the regulation of expression of components of the nucleic acid vector (e.g. , a nucleic acid sequence encoding a therapeutic molecule comprised therein).
  • compositions comprising a lipid enveloped rAAV particle as described herein further comprises a nucleic acid sequence encoding a promoter.
  • Promoters include, but are not limited to, constitutive promoters, inducible promoters, tissue- specific promoters, cell type-specific promoters, and synthetic promoters.
  • a composition comprising a lipid enveloped rAA V particle as described herein may include nucleic acid sequences encoding viral promoters or promoters from mammalian genes that are generally active in promoting transcription.
  • constitutive viral promoters include the Herpes Simplex virus (HSV), thymidine kinase (TK), Rous Sarcoma Virus (RSV), Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV), Ad E1A and cytomegalovirus (CMV) promoters.
  • constitutive mammalian promoters include various housekeeping gene promoters, as exemplified by the p-actin promoter.
  • Inducible promoters or other inducible regulatory elements may also be used to achieve desired expression levels of a therapeutic molecule (e.g., a protein or polypeptide of interest).
  • suitable inducible promoters include those from genes such as cytochrome P450 genes, heat shock protein genes, metallothionein genes, and hormone- inducible genes, such as the estrogen gene promoter.
  • Another example of an inducible promoter is the tetVP16 promoter that is responsive to tetracycline.
  • Tissue- and cell-specific promoters or other tissue- or cell-specific regulatory elements are also contemplated herein.
  • Synthetic promoters are also contemplated herein.
  • a synthetic promoter may comprise, for example, regions of known promoters, regulatory elements, transcription factor binding sites, enhancer elements, repressor elements, and the like.
  • the therapeutic molecule comprises a therapeutic or diagnostic protein or polypeptide.
  • a therapeutic or diagnostic protein or polypeptide is an antibody, a peptibody, a growth factor, a clotting factor, a hormone, a membrane protein, a cytokine, a chemokine, an activating or inhibitory peptide acting on cell surface receptors or ion channels, a cell-permeant peptide targeting intracellular processes, a thrombolytic agent, an enzyme, a bone morphogenetic protein, a nuclease, guide RNA or other nucleic acid or protein used for gene editing, an Fc-fusion protein, an anticoagulant, or a protein or polypeptide that can be detected using a laboratory test.
  • the therapeutic molecule comprises a gene therapy medication.
  • a gene therapy medication is used to treat spinal muscular atrophy.
  • a gene therapy medication used to treat spinal muscular atrophy is ona shogene abeparvovec .
  • a composition comprising a lipid enveloped rAAV particle as described herein may comprise a nucleic acid sequence which encodes a detectable molecule.
  • a detectable molecule is a molecule that can be visualized (e.g., using a naked eye, under a microscope, or using a light detection device such as a camera).
  • the detectable molecule is a fluorescent molecule, a bioluminescent molecule, or a molecule that provides color (e.g., ⁇ -galactosidase, p-lactamase, p-glucuronidase, or spheroidenone).
  • the detectable molecule is a fluorescent, bioluminescent or enzymatic protein or functional peptide or polypeptide thereof.
  • fluorescent protein is a blue fluorescent protein, a cyan fluorescent protein, a green fluorescent protein, a yellow fluorescent protein, an orange fluorescent protein, a red fluorescent protein, or a functional peptide or polypeptide thereof.
  • a blue fluorescent protein may be azurite, EBFP, EBFP2, mTagBFP, or Y66H.
  • a cyan fluorescent protein may be ECFP, AmCyanl, Cerulean, CyPet, mECFP, Midori-ishi Cyan, mTFPl, or TagCFP.
  • a Green fluorescent protein may be AcGFP, Azami Green, EGFP, Emarald, GFP or a mutated form of GFP (e.g., GFP-S65T, mWasabi, Slemmer, Superfolder GFP, TagGFP, TurboGFP, or ZsGreen).
  • a yellow fluorescent protein may be EYFP, mBanana, mCitrine, PhiYFp, TagYFP, Topaz, Venus, YPet, or Zs Yellowl .
  • An orange fluorescent protein may be DsRed, RFP, DsRed2, DsRed-Express, Ds-Red-monomer, Tomato, tdTomato, Kusabira Orange, mK02, mOrange, mOrange2, niTangerine, TagRFP, or TagRFP-T.
  • a red fluorescent protein may be AQ142, AsRed2, dKeima-Tandem, HcRedl , tHcRed, Jred, mApple, mCherry, mPlurn, mRaspberry, mRFPl , mRuby or mStrawberry.
  • a detectable molecule is a bioluminescent protein or a functional peptide or polypeptide thereof.
  • bioluminescent proteins are firefly luciferase, click-beetle luciferase, Renilla luciferase, and luciferase from Oplophorus gracilirostris .
  • a detectable molecule may be any polypeptide or protein that can be detected using methods known in the art. Non-limiting methods of detection are fluorescence imaging, luminescent imaging, bright field imaging, and imaging facilitated by immunofluorescence or immunohistochemical staining.
  • administering means providing a material to a subject in a manner that is pharmacologically useful.
  • the subject is a mammal.
  • the subject is a human, non-human primate, dog, cat, pig, mouse, horse, sheep, goat, rat, guinea pig, hamster, or cow.
  • the subject is a human.
  • the subject is a human child (e.g., a human being less than 18 years of age).
  • a lipid enveloped rAAV particle as described herein or a composition comprising a lipid enveloped rAA V particle as described herein is administered to a subject enterally.
  • an enteral administration of the lipid enveloped rAAV particle as described herein or the composition comprising a lipid enveloped rAAV particle as described herein is oral.
  • a lipid enveloped rAAV particle as described herein or a composition comprising a lipid enveloped rAAV particle as described herein is administered to the subject parenterally.
  • a lipid enveloped rAAV particle as described herein or a composition a lipid enveloped rAAV particle as described herein is administered to a subject subcutaneously, intraocularly, intravitreally, subretinally, intravenously (IV), intracerebro-ventricularly, intramuscularly, intrathecally (IT), intracistemally, intraperitoneally, via inhalation, topically, or by direct injection to one or more cells, tissues, or organs.
  • a lipid enveloped rAAV particle as described herein or a composition comprising a lipid enveloped rA AV particle as described herein is administered to the subject by injection into the hepatic artery or portal vein.
  • the lipid enveloped rA AV particle or the composition comprising the lipid enveloped rAAV particle is administered intramuscularly, intravenously, subcutaneously, intrathecally, intraperitoneally, or by direct injection into an organ or a tissue of the subject.
  • any lipid enveloped rAAV particles as disclosed herein may be comprised within a pharmaceutical composition comprising a pharmaceutically-acceptable carrier or may be comprised within a pharmaceutically-acceptable carrier.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the rAAV particle is comprised or administered to a subject.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum oil such as mineral oil, vegetable oil such as peanut oil, soybean oil, and sesame oil, animal oil, or oil of synthetic origin. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers.
  • Non-limiting examples of pharmaceutically acceptable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, polyacrylic acids, lubricating agents (such as talc, magnesium stearate, and mineral oil), wetting agents, emulsifying agents, suspending agents, preserving agents (such as methyl-, ethyl-, and propyl-hydroxy-benzoates), and pH adjusting agents (such as inorganic and organic acids and bases), and solutions or compositions thereof.
  • lubricating agents such as talc, magnesium stearate, and mineral oil
  • wetting agents such as talc, magnesium stearate, and mineral oil
  • carriers include phosphate buffered saline, HEPES -buffered saline, and water for injection, any of which may be optionally combined with one or more of calcium chloride dihydrate, disodium phosphate anhydrous, magnesium chloride hexahydrate, potassium chloride, potassium dihydrogen phosphate, sodium chloride, or sucrose.
  • saline e.g., sterilized, pyrogen-free saline
  • saline buffers e.g., citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer
  • amino acids e.g., citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer
  • amino acids e.g., citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer
  • amino acids urea
  • alcohols e.g., ascorbic acid
  • phospholipids e.g., proteins (for example, serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol.
  • USP grade carriers and excipients are particularly useful for delivery of lipid enveloped rAAV particles to human subjects.
  • compositions may contain at least about 0.1% of the therapeutic agent (e.g., lipid enveloped rAAV particle) or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation.
  • the amount of therapeutic agent(s) (e.g., lipid enveloped rAAV particle) in each therapeutically-useful composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound.
  • Factors such as solubi lity, bioavailability, biological half-life, route of administration, and product shelf life, as well as other pharmacological considerations, will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be designed.
  • a lipid enveloped rA AV particle as described herein or a composition comprising a lipid enveloped rAAV particle as described herein is administered to a subject to treat a disease or disorder.
  • To “treat” a disease or disorder means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.
  • the compositions described above or elsewhere herein are typically administered to a subject in an effective amount, that is, an amount capable of producing a desirable result. The desirable result will depend upon the active agent being administered.
  • an effective amount of lipid enveloped rAAV particles may be an amount of the particles that are capable of transferring an expression construct to a host organ, tissue, or cell.
  • a therapeutically acceptable amount may be an amount that is capable of treating a disease, e.g., a cancer.
  • dosage for any one subject depends on many factors, including the subject's size, body surface area, age, the particular composition to be administered, the active ingredient(s) in the composition, time and route of administration, general health, and other drags being administered concurrently.
  • the concentration of lipid enveloped rAAV particles administered to a subject may be on the order ranging from 10 6 to 10 14 particles/ml or 10 3 to 10 15 particles/ml, or any values therebetween for either range, such as for example, about 10 6 , 10', 10 8 , 10 9 , 10 30 , 10 11 , 10 12 , 10 3 , or 10 14 particles/ml.
  • lipid enveloped rAAV particles of a higher concentration than 10 31 particles/ml are administered.
  • the concentration of AAV particles administered to a subject may be on the order ranging from 10 6 to 10 34 vector genomes (vgs)/ml or 10 3 to 10 15 vgs/ml, or any values therebetween for either range (e.g., 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or 10 34 vgs/ml).
  • lipid enveloped rAAV particles of higher concentration than 10 13 vgs/ml are administered.
  • the lipid enveloped rAAV particles can be administered as a single dose, or divided into two or more administrations as may be required to achieve therapy of the particular disease or disorder being treated.
  • 0.0001 ml to 10 ml are delivered to a subject.
  • the number of lipid enveloped rAAV particles administered to a subject may be on the order ranging from 10 6 to 10 14 ‘ vgs/kg body mass of the subject, or any values therebetween (e.g., 10 6 , 10 7 , 10 s , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or 10 14 vgs/kg).
  • the dose of AAV particles administered to a subject may be on the order ranging from 10 11 to 10 15 vgs/kg.
  • the volume of a composition comprising a lipid enveloped rAAV particle delivered to a subject is 0.0001 ml to 10 ml.
  • a composition disclosed herein (e.g., comprising a lipid enveloped rAAV particle) is administered to a subject once.
  • the composition is administered to a subject multiple times (e.g., twice, three times, four times, five times, six times, or more).
  • Repeated administration to a subject may be conducted at a regular interval (e.g., daily, every other day, twice per week, weekly, twice per month, monthly, every six months, once per year, or less or more frequently) as necessary to treat (e.g., improve or alleviate) one or more symptoms of a disease, disorder, or condition in the subject.
  • the subject has or is suspected of having a disease or disorder that may be treated with gene therapy.
  • Example conditions for which rAAV-based gene therapy may find particular utility include, but are not limited to, cancer, diabetes, autoimmune disease, kidney disease, cardiovascular disease, pancreatic disease, intestinal disease, liver disease, neurological disease, neuromuscular disorder, neuromotor deficit, neuroskeletal impairment, neurological disability, neurosensory dysfunction, stroke, al -antitrypsin (AAT) deficiency, Batten’s disease, ischemia, an eating disorder, Alzheimer's disease, Huntington's disease, Parkinson's disease, skeletal disease and pulmonary disease.
  • AAT al -antitrypsin
  • the disease or disorder is selected from the group consisting of: Alpha 1 -Antitrypsin Deficiency, Phenylketonuria, Wilson Disease, Acute Intermittent Porphyria, Homozygous Familial Hypercholesterolemia, Crigler- Najjar Syndrome, ATTR Amyloidosis, Methylmalonic Acidemia, a Mucopolysaccharidosis, Glycogen Storage Disease Type la, Ornithine Transcarbamylase Deficiency, liver fibrosis, Hemophilia A, Hemophilia B, and Hemophilia C.
  • the disease or disorder is a cancer.
  • the cancer is one or more of: cancer of the buccal cavity or pharynx, cancer of the digestive tract, cancer of the colon, rectum, or anus, cancer of the respiratory tract, breast cancer, cancer of the cervix, uterus, vagina, or vulva, cancer of the uterine corpus or ovary, cancer of the male genital tract, cancer of the urinary tract, bone or soft tissue cancer, Kaposi sarcoma, melanoma of the skin, ocular melanoma, non-melanoma eye cancer, cancer of the brain and central nervous system, cancer of the thyroid and other endocrine glands, Hodgkin Lymphoma, Non-Hodgkin Lymphoma, myeloma, renal cancer, colorectal cancer, lung cancer, breast cancer, pancreatic cancer, prostate cancer, gastric cancer, GIST or glioblastoma.
  • a nucleic acid isolated or derived from the subject e.g., genomic DNA, mRNA, or cDNA from the subject
  • sequencing e.g., Sanger or next- generation sequencing
  • a mutation e.g., in a gene associated with a disease or disorder
  • administration of the lipid enveloped rAAV particles of the disclosure or compositions comprising the lipid enveloped rAAV particles of the disclosure results in the amelioration, prevention, and/or treatment of conditions such as peptide deficiency, polypeptide deficiency, peptide overexpression, polypeptide overexpression, including for example, conditions which result in diseases or disorders such as cancers, tumors, or other malignant growths, neurological deficit dysfunction, autoimmune diseases, articular diseases, cardiac or pulmonary diseases, ischemia, stroke, cerebrovascular accidents, transient ischemic attacks (TIA); diabetes and/or other diseases of the pancreas; cardiocirculatory disease or dysfunction (including, e.g., hypotension, hypertension, atherosclerosis, hypercholesterolemia, vascular damage or disease); neural diseases (including, e.g., Alzheimer's, Huntington's, Tay- Sach's and Parkinson's disease, memory loss, trauma, motor impairment, neuropathy, and related disorders): biliary, renal or
  • the method comprises contacting a cell wdth a lipid enveloped rAAV particle as described herein or a composition comprising a lipid enveloped rAAV particle as described herein.
  • a cell disclosed herein is a cell isolated or derived from a subject.
  • a cell is a mammalian cell (e.g., a cell isolated or derived from a mammal).
  • the cell is a human cell, non-human primate cell, rat cell, or mouse cell.
  • a cell is isolated or derived from a particular tissue of a subject, such as liver tissue.
  • the cell is a liver, brain, heart or retina cell.
  • a cell is a liver cell. In some embodiments, a cell is in vitro. In some embodiments, a cell is ex vivo. In some embodiments, a cell is in vivo. In some embodiments, a cell is within a subject (e.g., within a tissue or organ of a subject). In some embodiments, a cell is a primary cell. In some embodiments, a cell is from a cell line (e.g., an immortalized cell line). In some embodiments a cell is a cancer cell or an immortalized cell.
  • Methods of contacting a cell may comprise, for example, contacting a cell in a culture with a lipid enveloped rAAV particle as described herein or a composition comprising a lipid enveloped rA AV particle as described herein.
  • contacting a cell comprises adding a lipid enveloped rAAV particle as described herein or a composition comprising a lipid enveloped rAAV particle as described herein to the supernatant of a cell culture (e.g., a cell culture on a tissue culture plate or dish) or mixing a lipid enveloped rAAV particle as described herein or a composition comprising a lipid enveloped rAAV particle as described herein with a cell culture (e.g., a suspension cell culture).
  • a cell culture e.g., a suspension cell culture
  • contacting a cell comprises mixing a lipid enveloped rAAV particle as described herein or a composition comprising a lipid enveloped rAAV particle as described herein with another solution, such as a cell culture media, and incubating a cell with the mixture.
  • contacting a cell with a lipid enveloped rAAV particle as described herein or a composition comprising a lipid enveloped rAAV particle as described herein comprises administering a lipid enveloped rAAV particle as described herein or a composition comprising a lipid enveloped rAAV particle as described herein to a subject or device in which the cell is located.
  • contacting a cell comprises injecting a lipid enveloped rAAV particle as described herein or a composition comprising a lipid enveloped rAAV particle as described herein into a subject in which the cell is located.
  • contacting a cell comprises administering a lipid enveloped rAAV particle as described herein or a composition comprising a lipid enveloped rAAV particle as described herein directly to a cell, or into or substantially adjacent to a tissue of a subject in which the cell is present.
  • Embodiment 1 A composition comprising a plurality of rAAV particles, wherein at least about 20% of the rAAV particles are lipid enveloped rAAV particles.
  • Embodiment 2 The composition of embodiment 1, wherein at least about 25%?, at least about 30%, or at least about 35% of the rAAV particles are lipid enveloped rAAV particles.
  • Embodiment 3 The composition of embodiment 1 or embodiment 2, wherein at least about 10 11 , at least about 10 12 , at least about 10 13 at least about 10 14 , or at least about 10 15 of the rAAV particles are lipid enveloped rAAV particles.
  • Embodiment 4. The composition of any one of embodiments 1-3, wherein the AAV is AAV serotype 2 (AAV2).
  • Embodiment 5 The composition of any one of embodiments 1-3, wherein the AAV is AAV serotype 1 (AAV1), AAV serotype 3 (AAV3), AAV serotype 4 (A.AV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), AAV serotype 12 (AAV12), AAV serotype 13 (AAV13), AAV rhesus isolate 8 (AAVrh.8), AAV rhesus isolate 10 (AAVrh.10), AAV rhesus isolate 39 (AAVrh.39), or AAV rhesus isolate 74 (AAVrh.74).
  • AAV serotype 1 AAV1
  • AAV3 AAV serotype 3
  • Embodiment 6 The composition of any one of embodiments 1-5, further comprising a pharmaceutically-acceptable carrier or excipient.
  • Embodiment 7 The composition of embodiment 6, wherein the composition is sterilized.
  • Embodiment 8 A method of preparing a population of lipid enveloped rAAV particles comprising:
  • Embodiment 9 The method of embodiment 8, wherein the host producer cell is a mammalian or insect cell.
  • Embodiment 10 The method of embodiment 9, wherein the mammalian cell is a HEK293 cell, or wherein the insect cell is a Sf9 cell.
  • Embodiment 11 The method of any one of embodiments 8-10, further comprising a step of overexpressing membrane associated accessory protein (M AAP) and/or CD9 in the host producer cell.
  • M AAP membrane associated accessory protein
  • Embodiment 12 The method of embodiment 11, wherein both MAAP and CD9 are overexpressed.
  • Embodiment 13 The method of embodiment 11, wherein either MAAP or CD9 is overexpressed.
  • Embodiment 14 The method of any one of embodiments 8-13, wherein the harvested medium supernatant does not comprise non-lipid enveloped rAAV particles.
  • Embodiment 15 The method of any one of embodiments 8-14, wherein step (b) does not comprise harvesting the host producer cells.
  • Embodiment 16 The method of any one of embodiments 8-15, wherein following step (b), the method further comprises a step of adding fresh medium to the host producer cell, and re- harvesting the medium supernatant.
  • Embodiment 17 The method of any one of embodiments 8-16, wherein isolating the lipid enveloped rAAV particles comprises precipitating the lipid enveloped rAAV particles and/or purifying the precipitate.
  • Embodiment 18 The method of embodiment 17, wherein the lipid enveloped rAAV particles are precipitated using polyethylene glycol 8000 (PEG8000).
  • PEG8000 polyethylene glycol 8000
  • Embodiment 19 The method of embodiment 18, wherein the PEG8000 is in a concentration of at least about 5%, at least about 10%, or at least about 15% weight per volume (w/v).
  • Embodiment 20 The method of any one of embodiments 17-19, wherein purifying the precipitate comprises performing iodixanol gradient ultracentrifugation or sucrose-gradient ultracentrifugation.
  • Embodiment 21 The method of any one of embodiments 8-20, wherein at least about 20%, at least about 25%, at least about 30%, or at least about 35% of the isolated rAAV particles are lipid enveloped rAAV particles.
  • Embodiment 22 The method of any one of embodiments 8-21, wherein at least about 10 11 , at least about 10 12 , at least about 10 13 , at least about 10 14 , or at least about 10 15 of the isolated rAAV particles are lipid enveloped rAAV particles.
  • Embodiment 23 The method of any one of embodiments 8-22, wherein the AAV is AAV serotype 2 (AAV2).
  • AAV AAV serotype 2
  • Embodiment 24 The method of any one of embodiments 8-22, wherein the AAV is AAV serotype 1 (AAV1), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV 10), AAV serotype 11 (AAV 11), AAV serotype 12 (AAV12), AAV serotype 13 (AAV13), AAV rhesus isolate 8 (AAVrh.8), AAV rhesus isolate 10 (AAVrh.10), AAV rhesus isolate 39 (AAVrh.39), or AAV rhesus isolate 74 (AAVrh.74).
  • AAV serotype 1 AAV1
  • AAV3 AAV serotype 3
  • AAV serotype 4
  • Embodiment 25 A method of treating a disease or disorder comprising administering a composition according to any one of embodiments 1-7 to a subject, wherein the lipid enveloped rAAV particles in the composition comprise a nucleic acid encoding a therapeutic molecule.
  • Embodiment 26 The method of embodiment 25, wherein the disease or disorder is a cancer, diabetes, autoimmune disease, kidney disease, cardiovascular disease, pancreatic disease, intestinal disease, liver disease, neurological disease, neuromuscular disorder, neuromotor deficit, neuroskeletal impairment, neurological disability, neuroscnsory dysfunction, stroke, al- antitrypsin (AAT) deficiency, Batten's disease, ischemia, an eating disorder, Alzheimer’s disease, Huntington's disease, Parkinson's disease, skeletal disease, or pulmonary disease.
  • AAT al- antitrypsin
  • Embodiment 27 The method of embodiment 25 or embodiment 26, wherein the subject is a human subject.
  • Embodiment 28 The method of any one of embodiments 25-27, wherein the composition has enhanced immune evasion properties or is less immunogenic than a heterogenous composition of rAAV particles comprising non-lipid enveloped rAAV particles and/or vesicles or exosomes each comprising multiple rAAV particles.
  • Embodiment 29 The method of any one of embodiments 25-28, wherein the composition has greater infectivity than a heterogenous composition of rAAV particles comprising non-lipid enveloped rAAV particles and/or vesicles or exosomes each comprising multiple rAAV particles.
  • Embodiment 30 The method of any one of embodiments 25-29, wherein the composition has a greater multiplicity of infection (MOI) than a heterogenous composition of rAAV particles comprising non-lipid enveloped rAAV particles and/or vesicles or exosomes each comprising multiple rAAV particles.
  • MOI multiplicity of infection
  • Embodiment 31 The method of any one of embodiments 23-30, wherein the composition is administered to the subject one time or multiple times.
  • Embodiment 32 The composition of any one of embodiments 1-7, wherein the composition has enhanced immune evasion properties or is less immunogenic than a heterogenous composition of rAAV particles comprising non-lipid enveloped rAAV particles and/or vesicles or exosomes each comprising multiple rAAV particles.
  • Embodiment 33 The composition of any one of embodiments 1-7 or 32, wherein the composition has greater infectivity than a heterogenous composition of rAAV particles comprising non-lipid enveloped rAAV particles and/or vesicles or exosomes each comprising multiple rAAV particles.
  • Embodiment 34 The composition of any one of embodiments 1-7, 32, or 33, wherein the composition has a greater multiplicity of infection (MOI) than a heterogenous composition of rAAV particles comprising non-lipid enveloped rAAV particles and/or vesicles or exosomes each comprising multiple rAAV particles.
  • MOI multiplicity of infection
  • Embodiment 35 The method of any one of embodiments 8-24, wherein the heterogenous population of rAAV particles further comprises vesicles or exosomes each comprising multiple rAAV particles.
  • Embodiment 36 The method of embodiment 35, wherein the harvested medium supernatant does not comprise vesicles or exosomes each comprising multiple rAAV particles.
  • Embodiment 37 The method of embodiment 35, wherein the harvested medium supernatant does not comprise (i) non-lipid enveloped rAAV particles nor (ii) vesicles or exosomes each comprising multiple rAAV particles.
  • Embodiment 38 The composition of any one of embodiments 1-7 or the method of any one of embodiments 25-31, wherein the composition:
  • (d) has a greater multiplicity of infection (MOI), relative to a heterogenous composition of rAAV particles comprising non-lipid enveloped rAAV particles and/or vesicles or exosomes each comprising multiple rAAV particles.
  • MOI multiplicity of infection
  • Example 1 Characterization of lipid enveloped AAV particles
  • Adeno-associated viruses are classified as non-enveloped single stranded DNA (ssDNA) viruses belonging to the family Parvoviridae, genus Dependopan>ovirus.
  • the virus capsid is composed of the viral proteins (VPs) VP1 (83 kDa), VP2 (72 kDa), and VP3 (63 kDa) in a —1:1:10 ratio [2]
  • VP1 and VP2 are N-terminal extended forms of VP3, with the VP1 unique region (VPlu) encoding a phospholipase A2 (PLA2) domain which is required for infectivity [3]
  • the capsid structures of the AAV serotypes (AAV1 through AAV13) have been determined by either X-ray crystallography and/or cryo-electron micro
  • the AAV capsids are assembled from sixty VPs through 2-, 3-, and 5-fold VP interactions.
  • the capsid core consists of an anti-parallel, eight-stranded (pB to pl) p-barrel motif with an additional strand, ⁇ A, anti-parallel to ⁇ B and an a-helix (aA).
  • pB to pl anti-parallel, eight-stranded
  • aA anti-parallel to ⁇ B and an a-helix
  • aA a-helix
  • insertion loops form the exterior surface of the capsid. These are characterized by high amino acid sequence and structure variability.
  • Nine such regions at the apex of these insertion loops have been defined and assigned variable regions (VRs) by structural alignments [5, 6]. Despite these variabilities, the overall AAV capsid morphology is conserved between the serotypes.
  • All the AAV capsids possess cylindrical channels at the icosahedral 5-fold axes that are believed to be the route of genomic DNA packaging and VPlu extemalization following cell entry [7].
  • depressions are flanked by protrusions surrounding the 3-fold axes and raised regions between the 2- and 5-fold axes that are termed 2/5-fold walls.
  • the 2/5-fold wall and 3- fold protrusions have been identified as receptor binding sites for many AAV serotypes and play important roles in cell transduction [4], Additionally, these regions, including the 5-fold region, displays antigenic sites for antibodies raised by the host immune response [8].
  • AAVs are one of the most commonly used vectors for a variety of gene therapy applications.
  • two AAV biologies have been approved by the FDA [9, 10].
  • a major challenge for AAV-mediated gene therapy is the presence of pre-existing neutralizing antibodies against the AAV capsids in a large percentage of the population [11, 12], These antibodies can severely reduce the desired therapeutic effect or exclude patients from receiving an AAV-based gene therapy vector [11, 12] .
  • Strategies to reduce the antigenicity of the AAV capsids include the engineering of the capsid surface amino acids [13, 14] or the conjugation of molecules, e.g., polyethylene glycol [15, 16], to the surface of the capsid.
  • exosome-associated (enveloped) AAV exosome-associated AAV particles
  • Previous studies have shown that these exo-AAV are infectious and are currently being investigated in pre-clinical studies for gene therapy applications [17, 18, 19].
  • the capsids mediate the attachment to a cell receptor(s)
  • the mechanism of cell entry, trafficking, and uncoating for exo- A A Vs remain unclear.
  • the surrounding membrane shields the AAV capsids from neutralizing antibodies [19, 17].
  • the detailed mechanism of exosome-associated AAV infection remains unclear.
  • the exosome-associated AAV particles which have been observed thus far comprise multiple AAV particles per exosome.
  • MAAP membrane associated accessory protein
  • envelope-associated AAV2 (alternatively referred to herein as envelope-associated AAV2, or EA-AAV2), obtained from a Sf9 baculoviral expression system, and were able to determine their structure using cryo-electron microscopy (cryo-EM) at 3.14 A resolution.
  • the production and purification protocol described herein yielded exclusively singly-enveloped AAV2 comprising single AAV particles.
  • the size of the EA-AAV capsids was ⁇ 40 nm in diameter.
  • the 3-fold protrusions were observed to be located in close proximity to the membrane envelope.
  • the structure of the EA-AAV" capsid does not display observable structural differences from its non-enveloped AAV2 counterpart.
  • VLPs AAV2 virus-like particles
  • Sf9 baculoviral expression system comprising the entire nucleotide sequence of the AAV2 VP open reading frame (including the entire MAAP and AAP open reading frames within) driven by the polyhedron promoter was utilized.
  • the cell pellet was lysed by freeze-thawing and subsequently using a microfluidizer (Microfluidonics model LM- 10).
  • VLPs which were separated from cell lysate and PEG pellets were combined and purified by iodixanol gradient as previously described [30], and visualized by SDS-PAGE for confirmation of purity and VP protein composition.
  • Fractions containing VP1/2/3 in the 25 to 40% iodixanol fractions were combined and buffer exchanged using a 150 kDa cutoff concentrator (Orbital Biosciences, catalog #: AP0715010) into Universal Buffer pH 7.4 [31]. Sample purity and concentration were assessed by SDS-PAGE.
  • Negative stain electron microscopy was employed after purification to ensure the purity and integrity of the capsids in the sample. Once this was established, 5 ⁇ L of VLPs at 0.1 mg/mL were applied onto a glow-discharged copper grid (Electron Microscopy Supplies, catalog #: CFU-400Cu). The VLPs were incubated for 30 seconds before blotting. The grid was then washed 3 times for 10 seconds in filtered de-ionized water (>5 M ⁇ ), and then blotted dry again. The grid was then stained twice in 1% uranyl acetate for 5 seconds before blotting dry. Grids were imaged on an electron microscope (Thermo- Fisher model FE1 Spirit) operated at 120k V.
  • a nominal magnification of 81,000 x was used, giving a physical pixel size of 1.1 A (0.55 A per pixel at super-resolution mode).
  • Movies were recorded with a K3 camera operating in counting mode with dose rate of the electron beam set to 27 electrons per physical pixel per second on camera, corresponding to dosage of 22e-/A2/sec.
  • An accumulated dose of 34e- per A2 on the sample was fractionated into a movie stack of 30 image frames. Total 2,133 movies were recorded within eight-hour session with image shift using SerialEM software [32] . For each recorded movie, the frames were aligned for drift correction as previously described [33].
  • the final map was calculated using cisTEM flattened between 20-3.14 ⁇ (high resolution) or from 20-10 ⁇ (low resolution) [34] to either visualize the VLP structure or lipid envelope, respectively.
  • the maps were normalized using the Mapman software suite [35]. Both the non-enveloped and EA capsid maps have been deposited into EMDB with accession codes EMD-24719 and EMD-24718 respectively.
  • the atomic model was built and refined based the crystal structure of AAV2 (PDB: llp3) [ 1 ]. The structure was refined in Coot [36] and phenix [37] iteratively.
  • the atomic model for the non-enveloped and EA-AAV capsids have been deposited with the pdbid 7RWT & 7RWL respectively.
  • the particles selected for the non- enveloped and EA-AAV capsid were collected from the same micrographs and the final data statistics are given in Table 1.
  • VLPs virus like particles
  • SDS-PAGE gels of the purified VLPs showed VP1, VP2, and VP3 at their expected (1:1 :10) ratio with minor ( ⁇ 5%) impurities of proteins of >250 and -15-37 kDa molecular weights (data not shown).
  • negative stain EM the vast majority of capsids observed were non-enveloped.
  • a minor species of -40 nm diameter spheroids were observed which were stain impermeable in the preparation ( Figure 1A).
  • exosome size typically ranges from 40-100 nm in diameter
  • these particles were within the expected size range of exosomes [22]
  • a small number of 10-15 nm particles were also noted in the background likely representing impurities of presumably cellular proteins.
  • the 2D class classification of the boxed EA-AAV capsid particles resulted in two principal classes, identified from the final map as the near 2-fold (179 particles) ( Figures 2A(i) and 2B(i)) and near 5-fold (112 particles) ( Figures 2A(ii) and B(ii)) projections.
  • the envelope observed around the particle demonstrated an irregular halo of density with a repeating diffuse “zig-zag” pattern ring. This pattern is likely the result of noise resulting from mismatched symmetry between the capsid (icosahedral) and envelope (presumed non- symmetric). But it may also be signal from protein(s) embedded within the lipid. It is also noteworthy that the boundary limits of the inner radius of the envelope most likely contacts the AAV2 capsid 3-fold protrusions.
  • the envelope thickness was ⁇ 5 nm (Figure 3E), which is typical for a typical lipid bilayer [23, 24],
  • the cryo-EM map showed the closest approach of the capsid to the envelope at the 3 -fold protrusions with a distance of —5-10 A ( Figure 3F).
  • envelope density was “best” ordered around the 3 -fold axes and substantially less ordered over the 5-fold axes. Complete density coverage over the 3-fold was observed at 3 sigma map contouring, but near the 5-fold axes the map contour was 1 sigma to achieve coverage.
  • AAV capsids produced in Sf9 cells, contain a small but significant number of homogenous EA-AAV capsids (-1%).
  • the expression system used demonstrates that the generation of these particles is producer cell independent, and therefore allows for production of EA-AAV capsids by scalable production systems such as the Sf9 baculovirus system — though the ability to purify them is likely limited to means which exclude affinity base chromatography (e.g., anion exchange, or AAV- specific antibody/nanobody columns) [26, 27].
  • HAV Hepatitis A virus
  • Norovirus and Rotavirus utilize this strategy when shed from stool to infect the next host to successfully evade host proteases within the gut/stool.
  • the enveloped nature of these viruses allows them to achieve a higher multiple of infection in the next host [29]. It is believed that EA-AAVs may behave in a similar manner to these enteroviruses, as MAAP deletion variants are out competed by wtAAV2 by -10 fold margin [20], and in challenge to neutralizing antibodies in vivo retain higher infectivity [19, 17].
  • the EA-AAV capsids are ordered, and as such their structure could be determined at 3.14 A resolution.
  • the capsid structure has been determined to be identical to their non-enveloped AAV counterparts (2.43 A resolution).
  • the close contacts of the protrusions at the capsid 3-fold, along with polar and positive charge amino acid clusters, could imply that this region is involved in a non-specific interaction with the lipid envelope.
  • MAAP Given the expected role of MAAP in generating these small exosomes, it is possible that MAAP is present in a symmetry mis-matched manner, and hence not visualized. In this view, it is possible that MAAP is inducing both exosome formation and the subsequent recruitment of newly-produced AAV particles.
  • enveloped AAV particles likely utilizes host cell machinery for host cell uptake, even if MAAP alone is sufficient to induce envelope formation. If host machinery is required for EA-AAVs host cell uptake, but not required for production, this may provide a future roadmap for retargeting of capsids. Furthermore, the incorporation of proteins, peptides, and/or glycans into the EA-AAV membrane may aid in cell targeting. Therefore, it is hypothesized that MAAP-derived EA-AAV capsids may have therapeutic benefits of immune evasion and/or improved infectivity.
  • This example describes a protocol for the production of a heterogenous population of rAAV particles comprising non-lipid enveloped and lipid enveloped rAAV particles in a host producer cell comprised in a medium.
  • the heterogenous population may also, in some embodiments, comprise vesicles or exosomes each comprising multiple rAAV particles.
  • Bac-AAV2 Bac-AAV2 was amplified in Sf9 cells grown in suspension culture between 0.5-2 x 10 6 cells/mL growing in the log phase. Virus was titered by plaque assay in the same cells grown in confluent culture before amplification or infection. Cells were maintained in FBS serum free SF900II medium. Cells were not grown past passage 30.
  • Growth flasks for suspension culture were reusable glass Erlenmeyer flask with a minimum 5x excess head space (i.e., 50 mL culture grown in 250 mL), with the only exception being a 2.8 L flask used to grow at 1 L scale (Pyrex 4420). Plaque assays were carried out in 6 well plates, plated with 1 x 10 6 cells/ well.
  • amplification of Bac-AAV2 was carried out to a maximum of 3 passages in suspension culture, utilizing a multiple of infection (MOI) of 0.1, at a cell concentration of 2 x 10 6 .
  • MOI multiple of infection
  • EA capsids envelope-associated AAV2 capsids
  • 1 L cultures of Sf9 cells were infected with freshly titered Bac-AAV2 at an MOI of 5.
  • the cell pellet was separated from the PEG by centrifugation at 400 rcf (1.5k rpm) in a J A- 10 rotor for 20 minutes.
  • the cell pellet was aliquoted into 4 x 10 mL tubes of phosphate buffered saline supplemented with magnesium and potassium (TD).
  • TD buffer is used for downstream processing of Benzonase.
  • the supernatant was incubated in a final concentration of 10% w/v PEG8000 with 0.5M NaCl overnight at 4°C.
  • the solution was spun in a J A- 10 rotor at 14,000 ref (9k rpm) for 90 minutes at 4°C.
  • Upon stopping the supernatant was decanted and pellets were resuspended into 4 x 10 mL aliquots of TD.
  • One 10 mL tube is a single PEG pellet.
  • Cell pellet was 3x freeze thawed alternately between liquid nitrogen and 37°C, and then treated with 1 microliter of Benzonase (Sigma 71205-25KUN) for 1 hour at 37°C.
  • Cell pellet having 10% v/v 5M NaCl added then was spun in a J A -20 rotor at 8000rcf (8k rpm) for 10 minutes to clarify, the pellet was discarded. This was repeated until no pellet was formed.
  • cell and PEG pellets were purified by iodixanol, fractionated, and assessed by SDS-PAGE for VP content and purity. Pure fractions from the 25 to 40% iodixanol fractions were pooled. This was buffer exchanged into TD buffer for storage before data collection.
  • Example 3 Protocol for preparing a population of lipid enveloped rAAV particles
  • This example describes a protocol for preparing an enriched population of lipid enveloped rAAV particles.
  • the rAAV particles were produced as described in Examples 1 and 2. Following production, the medium supernatant is exclusively harvested. Harvesting of the producer cells is specifically avoided. Following removal of the medium supernatant, fresh medium is, in some embodiments, added to the producer cells, and subsequently re-harvested. Incorporating the step of adding fresh medium and re-harvesting is not necessary to the protocol; however, inclusion of this step allows for the potential to harvest lipid enveloped rAAV particle- containing medium supernatant multiple times from the same transfected or infected cells.
  • a step of enrichment is optionally performed wherein membrane associated accessory protein (MAAP) and/or CD9 protein is overexpressed in the host producer cells.
  • MAAP membrane associated accessory protein
  • CD9 may be overexpressed individually, or both MAAP and CD9 may both be overexpressed in combination.
  • Overexpression of MAAP and/or CD9 enhances the production of lipid enveloped rAAV particles in the host producer cells, and/or may facilitate the release of lipid enveloped rAAV particles from the host cells into the medium supernatant.
  • the lipid enveloped rAAV particles are precipitated using 10% w/v PEG8000. Following precipitation, the precipitate is purified using iodixanol gradient ultracentrifugation, or, alternatively, sucrose-gradient ultracentrifugation.
  • Example 4 Method of treating a disease or disorder via administration of a composition comprising lipid enveloped rAAV partides comprising a therapeutic molecule
  • This example describes a protocol for treating a disease or disorder by administering a composition comprising lipid enveloped rAAV particles (as described herein) to a subject.
  • the lipid enveloped rAAV particles are produced as described in Examples 1-3.
  • a therapeutic molecule e.g., transgene
  • the therapeutic molecule may, for example, be a gene therapy medication used to treat spinal muscular atrophy (e.g., onathiogene abeparvovec).
  • spinal muscular atrophy e.g., onacriogene abeparvovec
  • other therapeutic molecules are specifically contemplated herein.
  • the lipid envelope surrounding the rAAV capsids is obtained after genome packaging, at the time when the capsids are released from the producer cells.
  • composition comprising the lipid enveloped rAAV particles comprising a therapeutic molecule is administered to a subject having a disease or disorder.
  • the therapeutic molecule is a gene therapy medication used to treat spinal muscular atrophy (e.g., onahimogene abeparvovec)
  • the disease or disorder is spinal muscular atrophy
  • the method of administration is a single dose via direct injection into a vein of the subject (e.g., an IV infusion over 60 minutes), and the dosage is, for example, 1.1 x 10 14 vector genomes (vg) per kg of body weight.
  • vg vector genomes
  • other diseases and disorders, other routes of administration, and other dosages are specifically contemplated herein.
  • one or more signs or symptoms of the disease or disorder is alleviated or ameliorated.
  • the therapeutic molecule is a gene therapy medication used to treat spinal muscular atrophy (e.g., ona shogene abeparvovec)
  • administration of the composition may, for example, slow or halt the progression of spinal muscular atrophy, and/or may result in decreased muscle weakness, increased muscle tone, increased mobility, mitigation or alleviation of breathing problems, mitigation or alle viation of problems eating and swallowing, reduction of spontaneous tongue movements, and/or reduction in scoliosis (curvature of the spine).
  • inventive embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed.
  • inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein.
  • a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified.
  • “at least one of A and B” can refer, in one embodiment, to at least one, optionally including more than one. A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one. A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
  • composition comprising A and B
  • composition also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B”.

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Abstract

La divulgation concerne des particules d'AAVr enveloppées de lipides, des compositions les comprenant, et des procédés de traitement d'une maladie comprenant l'administration de tels peptides d'AAVr enveloppés de lipides et de telles compositions. La divulgation concerne également des procédés de préparation de populations de particules d'AAVr enveloppées de lipides. L'AAV peut être un AVV de sérotype 1 (AAV1), et la composition comprend un véhicule ou un excipient pharmaceutiquement acceptable.
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