METHODS AND COMPOSITIONS FOR MODIFYING ASSEMBLY-ACTIVATING PROTEIN (AAP)-DEPENDENCE OF VIRUSES
1. An adeno-associated virus (AAV) capsid polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:
Methods and compositions are provided that can be used to modify the assembly activating protein (AAP)-dependence of an adeno-associated virus (AAV).
- 1. An adeno-associated virus (AAV) capsid polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:
- 7. A nucleic acid molecule comprising a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:
- 4 and encoding an adeno-associated virus (AAV) capsid polypeptide.
- View Dependent Claims (8, 9, 10, 11, 12, 13)
- 23. An AAV capsid polypeptide comprising the amino acid sequence of SEQ ID NO:
- 1 and further comprising the amino acid residues at the indicated positions shown in Table 1 for AAP-dependence.
This application is a continuation of International Patent Application No. PCT/US2018/032166, filed on May 10, 2018, which claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Application No. 62/669,901, filed on May 10, 2018, and U.S. Application No. 62/504,318, filed on May 10, 2017, all three of which are incorporated herein by reference in their entirety.
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 10, 2019, is named Sequence Listine.txt and is 19,481 bytes in size.
This disclosure generally relates to viral vector systems.
Adeno-associated virus (AAV) is a leading platform in therapeutic gene transfer, primarily for in vivo gene therapy approaches. While preclinical and clinical studies continue to demonstrate AAV'"'"'s potential as a reagent for safe and efficient gene delivery to alleviate a number of diseases, a bottleneck to its broader application is the production of sufficient vector quantities to treat these patient populations.
In general, this disclosure describes and demonstrates the utility of a particular sequence motif within an AAV capsid protein that enables the assembly-activating protein (AAP)-dependence of the AAV to be modified. Thus, this sequence motif can be used to address and alleviate at least one of the bottlenecks encountered in the production of virus vectors. In particular, this disclosure describes a minimal motif defined through a novel phenotype-to-phylogeny mapping method that can be used to modify the AAP-dependence of a virus. Briefly, a number of ancestral AAVs that have been developed (see, for example, WO 2015/054653 and WO 2017/019994, which are incorporated herein by reference in their entirety) were used to examine AAP dependence across a wide structural differential. This analysis allowed for the identification of a minimal motif that determines AAP dependency.
In one aspect, the disclosure features adeno-associated virus (AAV) capsid polypeptides including an amino acid sequence having at least 95% sequence identity (e.g., at least 99% sequence identity) to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the AAV capsid polypeptide has the amino acid sequence of SEQ ID NO:3. In some embodiments, the AAV capsid polypeptides are encoded by the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the AAV capsid polypeptide has the amino acid sequence of SEQ ID NO: 1, but contains the amino acid residues at the indicated positions shown in Table 1 for “independence” or “dependence” with respect to AAP.
This disclosure also features virus particles including any of the adeno-associated virus (AAV) capsid polypeptides described herein. Such virus particles can further include a transgene.
In another aspect, the disclosure features nucleic acid molecules including a nucleic acid sequence having at least 95% sequence identity (e.g., at least 99% sequence identity) to the nucleic acid sequence of SEQ ID NO: 4 and encoding an adeno-associated virus (AAV) capsid polypeptide. In some embodiments, the nucleic acid molecule has the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the nucleic acid molecule encodes the amino acid sequence of SEQ ID NO: 3.
The disclosure also provides vectors including any of the nucleic acid molecules described herein, as well as host cells including any of the nucleic acid molecules and/or vectors described herein. In some embodiments, the host cell is a packaging cell.
In another aspect, the disclosure features packaging cells including a nucleic acid molecule encoding an adeno-associated virus (AAV) capsid polypeptide, wherein the AAV capsid polypeptide has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the packaging cell lacks the assembly activating protein (AAP).
In another aspect, the disclosure includes methods of reducing the assembly activating protein (AAP)-dependence of an adeno-associated virus (AAV). Such methods include providing an AAV having a capsid polypeptide that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 3.
In yet another aspect, the disclosure features methods of relieving, at least partially, the assembly activating protein (AAP)-dependence of an adeno-associated virus (AAV), the method including: incorporating a capsid polypeptide into the AAV that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 3.
In still another aspect, the disclosure provides methods of engineering an adeno-associated virus (AAV) to reduce its dependence on assembly activating protein (AAP), including: engineering an AAV that comprises a capsid polypeptide that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 3.
Any of the methods described herein can further include culturing the adeno-associated virus (AAV) in the absence of the assembly activating protein (AAP). Any of the methods described herein further can include sequencing the engineered adeno-associated virus (AAV). Any of the methods described herein further can include comparing the assembly activating protein (AAP)-dependence of the engineered adeno-associated virus (AAV) relative to a non-engineered or wild type AAV. Any of the methods described herein further can include aligning the engineered adeno-associated virus (AAV) with the non-engineered or wild type AAV.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions of matter belong. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of matter, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Gene transfer, either for experimental or therapeutic purposes, relies upon a vector or vector system to shuttle genetic information into target cells. The vector or vector system is considered the major determinant of efficiency, specificity, host response, pharmacology, and longevity of the gene transfer reaction. Currently, the most efficient and effective way to accomplish gene transfer is through the use of vectors or vector systems based on viruses that have been made replication-defective. One of the most common viruses to be made replication-defective and used in gene transfer is adeno-associated virus (AAV).
The AAV capsid is a non-enveloped, icosahedral 60-mer of three repeating protein monomer subunits called viral protein 1 (VP1), VP2, and VP3. A single transcript expressed from the AAV cap gene containing nested open reading frames (ORFs) is alternately spliced, resulting in three distinct protein products that share C-terminal identity the length of VP3. A 1:1:10 stoichiometry of VP1:VP2:VP3 in the assembled capsid is thought to be a consequence of the relative abundance of each protein, which is, in turn, regulated by splice product abundance and a non-canonical ACG translation start codon for VP2.
The Assembly-Activating Protein (AAP) is a non-structural protein expressed from a non-canonical CTG start codon of an overlapping reading frame embedded within the capsid (cap) gene of AAV. AAV serotypes have different requirements for AAP, with some AAV serotypes exhibiting AAP-dependence (e.g., AAV8, rh10, Anc80, Anc81, Anc82, Anc83, and Anc84) and other AAV serotypes exhibiting AAP-independence (e.g., AAV9, rh8, and Anc110).
As used herein, an ancestral scaffold sequence refers to a non-naturally occurring sequence that is constructed using evolutionary probabilities and evolutionary modeling and is not known to have ever existed or to presently exit in nature. These scaffold sequences were leveraged herein to interrogate AAP function and delineate structural determinants within the capsid relevant to the virus'"'"' requirement for AAP.
This disclosure provides methods of modifying the AAP-dependence of an AAV. For example, an AAV capsid sequence can be engineered to include the motif identified herein, which reduces the AAP-dependence (or, conversely, increases the AAP-independence) during packaging of the AAV. This provides a number of benefits during manufacturing including, without limitation, the ability to reduce the number of components needed for productive particle assembly in any AAV production system (e.g. mammalian, yeast, insect cell), the ability to optimize AAV capsid structure with reduced constraints imposed by AAP, the potential of AAV capsid self assembly from minimal components, and the reduction of AAP contamination concerns in the final vector preparations.
A non-naturally occurring AAV capsid sequence, based originally on the Anc82 sequence (SEQ ID NO: 1, encoded by SEQ ID NO:2, both shown below), which exhibits AAP-dependence during packaging, has been modified as described herein to produce Anc82DI (SEQ ID NO:3, encoded by SEQ ID NO:4, both shown below). Anc82DI exhibits AAP-independence during packaging, but appears to retain functionality as a potent gene transfer vector. The sequence motif that imparts AAP-independence on AAP-dependent sequences is provided in Table 1 above.
In addition to the polypeptides having the amino acid sequences shown in SEQ ID NOs: 1 and 3, polypeptides are provided that have at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity) to the 55 polypeptides having the amino acid sequences shown in SEQ ID NOs: 1 and 3.
Similarly, nucleic acid molecules are provided that have at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity) to the nucleic acid molecules shown in SEQ ID NOs: 2 and 4.
In calculating percent sequence identity, two sequences are aligned and the number of identical matches of nucleotides or amino acid residues between the two sequences is determined. The number of identical matches is divided by the length of the aligned region (i.e., the number of aligned nucleotides or amino acid residues) and multiplied by 100 to arrive at a percent sequence identity value. It will be appreciated that the length of the aligned region can be a portion of one or both sequences up to the full-length size of the shortest sequence. It also will be appreciated that a single sequence can align with more than one other sequence and hence, can have different percent sequence identity values over each aligned region.
The alignment of two or more sequences to determine percent sequence identity can be performed using the algorithm described by Altschul et al. (1997, Nucleic Acids Res., 25:3389 3402) as incorporated into BLAST (basic local alignment search tool) programs, available at ncbi.nlm.nih.gov on the World Wide Web. BLAST searches can be performed to determine percent sequence identity between a sequence (nucleic acid or amino acid) and any other sequence or portion thereof aligned using the Altschul et al. algorithm. BLASTN is the program used to align and compare the identity between nucleic acid sequences, while BLASTP is the program used to align and compare the identity between amino acid sequences. When utilizing BLAST programs to calculate the percent identity between a sequence and another sequence, the default parameters of the respective programs generally are used.
This disclosure also provides vectors containing nucleic acid molecules that encode polypeptides. Vectors, including expression vectors, are commercially available or can be produced by recombinant technology. A vector containing a nucleic acid molecule can have one or more elements for expression operably linked to such a nucleic acid molecule, and further can include sequences such as those encoding a selectable marker (e.g., an antibiotic resistance gene), and/or those that can be used in purification of a polypeptide (e.g., 6×His tag). Elements for expression include nucleic acid sequences that direct and regulate expression of nucleic acid coding sequences. One example of an expression element is a promoter sequence. Expression elements also can include one or more of introns, enhancer sequences, response elements, or inducible elements that modulate expression of a nucleic acid molecule. Expression elements can be of bacterial, yeast, insect, mammalian, or viral origin and vectors can contain a combination of expression elements from different origins. As used herein, operably linked means that elements for expression are positioned in a vector relative to a coding sequence in such a way as to direct or regulate expression of the coding sequence.
A nucleic acid molecule, e.g., a nucleic acid molecule in a vector (e.g., an expression vector, such as a viral vector) can be introduced into a host cell. The term “host cell” refers not only to the particular cell(s) into which the nucleic acid molecule has been introduced, but also to the progeny or potential progeny of such a cell. Many suitable host cells are known to those skilled in the art; host cells can be prokaryotic cells (e.g., E. coli) or eukaryotic cells (e.g., yeast cells, insect cells, plant cells, mammalian cells). Representative host cells can include, without limitation, A549, WEHI, 3T3, 10T½, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, 293 cells, Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblast, hepatocyte and myoblast cells derived from mammals including human, monkey, mouse, rat, rabbit, and hamster. Methods for introducing nucleic acid molecules into host cells are well known in the art and include, without limitation, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and viral-mediated nucleic acid transfer (e.g., transduction).
With respect to polypeptides, “purified” refers to a polypeptide (i.e., a peptide or a polypeptide) that has been separated or purified from cellular components that naturally accompany it. Typically, the polypeptide is considered “purified” when it is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or 99%) by dry weight, free from the polypeptides and naturally occurring molecules with which it is naturally associated. Since a polypeptide that is chemically synthesized is, by nature, separated from the components that naturally accompany it, a synthetic polypeptide is considered “purified,” but further can be removed from the components used to synthesize the polypeptide (e.g., amino acid residues). With respect to nucleic acid molecules, “isolated” refers to a nucleic acid molecule that is separated from other nucleic acid molecules that are usually associated with it in the genome. In addition, an isolated nucleic acid molecule can include an engineered nucleic acid molecule such as a recombinant or a synthetic nucleic acid molecule.
Polypeptides can be obtained (e.g., purified) from natural sources (e.g., a biological sample) by known methods such as DEAE ion exchange, gel filtration, and/or hydroxyapatite chromatography. A purified polypeptide also can be obtained, for example, by expressing a nucleic acid molecule in an expression vector or by chemical synthesis. The extent of purity of a polypeptide can be measured using any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. Similarly, nucleic acid molecules can be obtained (e.g., isolated) using routine methods such as, without limitation, recombinant nucleic acid technology (e.g., restriction enzyme digestion and ligation) or the polymerase chain reaction (PCR; see, for example, PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995). In addition, isolated nucleic acid molecules can be chemically synthesized.
After the desired sequence of a virus or portion thereof has been determined (e.g., having a modified AAP-dependency), the actual nucleic acid molecule and/or polypeptide(s) can be generated, e.g., synthesized. Methods of generating an artificial nucleic acid molecule or polypeptide based on a sequence obtained, for example, in silico, are known in the art and include, for example, chemical synthesis or recombinant cloning. Additional methods for generating nucleic acid molecules or polypeptides are known in the art and are discussed in more detail below.
Once a polypeptide has been produced, or once a nucleic acid molecule has been generated and expressed to produce a polypeptide, the polypeptide can be assembled into a virus particle using, for example, a packaging host cell. The components of a virus particle (e.g., rep sequences, cap sequences, inverted terminal repeat (ITR) sequences) can be introduced, transiently or stably, into a packaging host cell using one or more vectors as described herein.
Virus particles can be purified using routine methods. As used herein, “purified” virus particles refer to virus particles that are removed from components in the mixture in which they were made such as, but not limited to, viral components (e.g., rep sequences, cap sequences), packaging host cells, and partially- or incompletely-assembled virus particles.
Once assembled, the virus particles can be screened for, e.g., the ability to replicate; gene transfer properties; receptor binding ability; and/or seroprevalence in a population (e.g., a human population). Determining whether a virus particle can replicate is routine in the art and typically includes infecting a host cell with an amount of virus particles and determining if the virus particles increase in number over time. Determining whether a virus particle is capable of performing gene transfer also is routine in the art and typically includes infecting host cells with virus particles containing a transgene (e.g., a detectable transgene such as a reporter gene, discussed in more detail below). Following infection and clearance of the virus, the host cells can be evaluated for the presence or absence of the transgene. Determining whether a virus particle binds to its receptor is routine in the art, and such methods can be performed in vitro or in vivo.
Determining the seroprevalence of a virus particle is routinely performed in the art and typically includes using an immunoassay to determine the prevalence of one or more antibodies in samples (e.g., blood samples) from a particular population of individuals. Seroprevalence is understood in the art to refer to the proportion of subjects in a population that is seropositive (i.e., has been exposed to a particular pathogen or immunogen), and is calculated as the number of subjects in a population who produce an antibody against a particular pathogen or immunogen divided by the total number of individuals in the population examined. Immunoassays are well known in the art and include, without limitation, an immunodot, Western blot, enzyme immunoassays (EIA), enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA). Simply by way of example, see Xu et al. (2007, Am. J. Obstet. Gynecol., 196:43.e1-6); Paul et al. (1994, J. Infect. Dis., 169:801-6); Sauerbrei et al. (2011, Eurosurv., 16(44):3); Boutin et al. (2010, Hum. Gene Ther., 21:704-12); Calcedo et al. (2009, J. Infect. Dis., 199:381-90); and Sakhria et al. (2013, PLoS Negl. Trop. Dis., 7:e2429), each of which determined seroprevalence for a particular antibody in a given population.
As described herein, virus particles can be neutralized by a person'"'"'s, e.g., patient'"'"'s, immune system. Several methods to determine the extent of neutralizing antibodies in a serum sample are available. For example, a neutralizing antibody assay measures the titer at which an experimental sample contains an antibody concentration that neutralizes infection by 50% or more as compared to a control sample without antibody. See, also, Fisher et al. (1997, Nature Med., 3:306-12) and Manning et al. (1998, Human Gene Ther., 9:477-85).
A virus or portion thereof that has a modified AAP-dependence as described herein can be used in a number of research and/or therapeutic applications. For example, a virus or portion thereof that has a modified AAP-dependence as described herein can be used in human or animal medicine for gene therapy (e.g., in a vector or vector system for gene transfer) or for vaccination (e.g., for antigen presentation). More specifically, a virus or portion thereof that has a modified AAP-dependence as described herein can be used for gene addition, gene augmentation, genetic delivery of a polypeptide therapeutic, genetic vaccination, gene silencing, genome editing, gene therapy, RNAi delivery, cDNA delivery, mRNA delivery, miRNA delivery, miRNA sponging, genetic immunization, optogenetic gene therapy, transgenesis, DNA vaccination, or DNA immunization.
A host cell can be transduced or infected with a virus or portion thereof having a modified AAP-dependence in vitro (e.g., growing in culture) or in vivo (e.g., in a subject). Host cells that can be transduced or infected with a virus or portion thereof having a modified AAP-dependence in vitro are described herein; host cells that can be transduced or infected with an ancestral virus or portion thereof in vivo include, without limitation, brain, liver, muscle, lung, eye (e.g., retina, retinal pigment epithelium), kidney, heart, gonads (e.g., testes, uterus, ovaries), skin, nasal passages, digestive system, pancreas, islet cells, neurons, lymphocytes, ear (e.g., inner ear), hair follicles, and/or glands (e.g., thyroid).
A virus or portion thereof having a modified AAP-dependence as described herein can be modified to include a transgene (in cis or trans with other viral sequences). A transgene can be, for example, a reporter gene (e.g., beta-lactamase, beta-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent polypeptide (GFP), chloramphenicol acetyltransferase (CAT), or luciferase, or fusion polypeptides that include an antigen tag domain such as hemagglutinin or Myc) or a therapeutic gene (e.g., genes encoding hormones or receptors thereof, growth factors or receptors thereof, differentiation factors or receptors thereof, immune system regulators (e.g., cytokines and interleukins) or receptors thereof, enzymes, RNAs (e.g., inhibitory RNAs or catalytic RNAs), or target antigens (e.g., oncogenic antigens, autoimmune antigens)).
The particular transgene will depend, at least in part, on the particular disease or deficiency being treated. Simply by way of example, gene transfer or gene therapy can be applied to the treatment of hemophilia, retinitis pigmentosa, cystic fibrosis, leber congenital amaurosis, lysosomal storage disorders, inborn errors of metabolism (e.g., inborn errors of amino acid metabolism including phenylketonuria, inborn errors of organic acid metabolism including propionic academia, inborn errors of fatty acid metabolism including medium-chain acyl-CoA dehydrogenase deficiency (MCAD)), cancer, achromatopsia, cone-rod dystrophies, macular degenerations (e.g., age-related macular degeneration), lipopolypeptide lipase deficiency, familial hypercholesterolemia, spinal muscular atrophy, Duchenne'"'"'s muscular dystrophy, Alzheimer'"'"'s disease, Parkinson'"'"'s disease, obesity, inflammatory bowel disorder, diabetes, congestive heart failure, hypercholesterolemia, hearing loss, coronary heart disease, familial renal amyloidosis, Marfan'"'"'s syndrome, fatal familial insomnia, Creutzfeldt-Jakob disease, sickle-cell disease, Huntington'"'"'s disease, fronto-temporal lobar degeneration, Usher syndrome, lactose intolerance, lipid storage disorders (e.g., Niemann-Pick disease, type C), Batten disease, choroideremia, glycogen storage disease type II (Pompe disease), ataxia telangiectasia (Louis-Bar syndrome), congenital hypothyroidism, severe combined immunodeficiency (SCID), and/or amyotrophic lateral sclerosis (ALS).
A transgene also can be, for example, an immunogen that is useful for immunizing a subject (e.g., a human, an animal (e.g., a companion animal, a farm animal, an endangered animal). For example, immunogens can be obtained from an organism (e.g., a pathogenic organism) or an immunogenic portion or component thereof (e.g., a toxin polypeptide or a by-product thereof). By way of example, pathogenic organisms from which immunogenic polypeptides can be obtained include viruses (e.g., picomavirus, enteroviruses, orthomyxovirus, reovirus, retrovirus), prokaryotes (e.g., Pneumococci, Staphylococci, Listeria, Pseudomonas), and eukaryotes (e.g., amebiasis, malaria, leishmaniasis, nematodes). It would be understood that the methods described herein and compositions produced by such methods are not to be limited by any particular transgene.
A virus or portion thereof having a modified AAP-dependence, usually suspended in a physiologically compatible carrier, can be administered to a subject (e.g., a human or non-human mammal). Suitable carriers include saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline), lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, and water. The virus or portion thereof having a modified AAP-dependence is administered in sufficient amounts to transduce or infect the cells and to provide sufficient levels of gene transfer and expression to provide a therapeutic benefit without undue adverse effects. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to an organ such as, for example, the liver or lung, orally, intranasally, intratracheally, by inhalation, intravenously, intramuscularly, intraocularly, subcutaneously, intradermally, transmucosally, or by other routes of administration. Routes of administration can be combined, if desired.
The dose of the virus or portion thereof having a modified AAP-dependence that is administered to a subject will depend primarily on factors such as the condition being treated, and the age, weight, and health of the subject. For example, a therapeutically effective dosage of a virus or portion thereof having a modified AAP-dependence that is to be administered to a human subject generally is in the range of from about 0.1 ml to about 10 ml of a solution containing concentrations of from about 1×101 to 1×1012 genome copies (GCs) of viruses (e.g., about 1×103 to 1×109 GCs). Transduction and/or expression of a transgene can be monitored at various time points following administration by DNA, RNA, or protein assays. In some instances, the levels of expression of the transgene can be monitored to determine the frequency and/or amount of dosage. Dosage regimens similar to those described for therapeutic purposes also may be utilized for immunization.
In accordance with the present invention, there may be employed conventional molecular biology, microbiology, biochemical, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. The invention will be further described in the following examples, which do not limit the scope of the methods and compositions of matter described in the claims.
Adeno-associated viral vectors were pseudotyped with either extant or ancestral viral capsids. Extant capsids include AAV1 (Genbank [GB] AAD27757.1), AAV2 (GB AAC03780.1), AAV3 (GB U48704.1), AAV4 (GB U89790.1), AAV5 (GB AAD13756.1), AAV6 (GB AF028704.1), AAV7 (NC_006260.1) Rh.10 (gb AA088201.1), AAV8 (GB AAN03857.1), AAV9 (GB AAS99264.1), and Rh32.33 (GB EU368926). Ancestral AAV capsids include Anc80L65, Anc81, Anc82, Anc83, Anc84, Anc110, Anc113, Anc126, and Anc127 (KT235804-KT235812). In this study, Anc83 has the following mutation in the presumed AAP ORF: Q1L (83AAP-KI).
AAPstop60, AAPstop20, and 82DI single revertant mutations were generated using the QuikChange® II Site-Directed Mutagenesis Kit according to the manufacturer'"'"'s instructions. To generate 82DI, the QuikChange® Lightning Multi Site-Directed Mutagenesis Kit was used according to the manufacturer'"'"'s instructions, in two phases: first, five sites were mutated on an Anc82 backbone, then the remaining five mutations were introduced into this quintuple mutant backbone.
Virus preparations to assay production in all serotypes and mutants were prepared as follows: Polyethylenimine transfections of AAV cis ITR-CMV-EGFP-T2A-Luc-ITR (2 μg), AAV trans rep-cap (2 μg), and adenovirus helper plasmid (4 μg) were performed on HEK 293 cells at 90% confluency in 6-well dishes. PEI Max (Polysciences)/DNA ratio was maintained at 1.375:1 (w/w) in serum-free media. Virus was harvested after 72 h by three freeze/thaw cycles followed by centrifugation at 15000×g.
For DRP titers, crude preps were DNaseI treated, and resistant (packaged) vector genome copies were used to titrate preps by TaqMan qPCR amplification (Applied Biosystems 7500, Life Technologies) with primers and probes detecting CMV promoter regions of the transgene cassette.
Thermostability of purified vector was assayed by AAV-ID (Pacouret et al., 2017, Mol. Ther., 25:1375-86). Briefly, A 500 uL sample of SYPRO® Orange 50X was prepared using PBS2+ (21-030-CV, Corning Inc., Corning, N.Y.) as a solvent. 96-well plates were loaded with 45 uL samples, supplemented with 5 uL Sypro Orange 50X. PBS2+ and 0.25 mg/mL Lysozyme (L6876, SIGMA-ALDRICH, St. Louis, Mo., USA) solutions were used as negative and positive controls, respectively. Plates were sealed and centrifuged at 3000 rpm for 2 min, and subsequently loaded into a 7500 Real-Time PCR System (ThermoFisher SCIENTIFIC). Samples were incubated at 25° C. for 2 min prior to undergo a temperature gradient (25 to 99° C., ˜2° C./10 min, step and hold mode with 0.4° C. temperature increments), while monitoring the fluorescence of the SYPRO® Orange dye using the ROX filter cube available on both qPCR systems. Fluorescence signals F were normalized between 0 and 100% and melting temperatures were defined as the temperature for which the numerical derivative dF/dT reached its maximum.
A20 capsid ELISAs were performed on crude virus preparations with the PROGEN AAV 2 Titration ELISA kit (ref# PRATV), according to the manufacturer'"'"'s instructions.
hA1AT ELISAs were performed on 1:1250-1:10000 serial dilutions of mouse serum with the Cloud-Clone ELISA kit for a-1 antitrypsin (SEB697Hu, 96 tests) according to the manufacturer'"'"'s instructions.
C57BL/6 male mice (6-8 weeks) were purchased from Jackson Laboratories. All experimental procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Schepens Eye Research Institute.
Mice were anesthetized with Ketamine/Xylazine intraperitoneally. Each animal was injected retro-orbitally (100 μl) with 1.00E+11 VG/mouse of the following vectors: Anc82.CB7.CI.EGFP.FF2A.hA1AT.RBG and Anc82DI.CB7.CI.EGFP.FF2A.hA1AT.RBG. Blood was collected via submandibular bleeds using GoldenRod animal lancets (MEDIpoint, Inc.) prior injection, and 3, 7, 15 and 28 days post injection. Samples were centrifuged at 8,000 rpm for 7.5 minutes and the serum was collected.
Animals were euthanized, and livers were collected and submerged in 4% paraformaldehyde solution (Electron Microscopy Sciences) for 30 minutes, then placed in 30% sucrose overnight. The next day the liver was mounted in Tissue-Tek O.C.T. Compound (Sakura Finetek) and flash frozen in cool isopentane.
To visualize eGFP expression in liver, 15 μm sections were mounted with VECTASHIELD® Hard Set™ mounting medium with DAPI (H-1500) and imaged with a Zeiss Axio Imager M2, at same gain and intensity across all sections.
All molecular representations in this study were generated using PyMOL and Protein Data Bank files 2QA0 (AAV8) and 3UX1 (AAV9).
Vectors were purified by affinity chromatography using either AVB Sepharose HP (25-4112-11, GE Healthcare) (AAV3 and AAV3s) or POROS CaptureSelect AAV9 affinity resin (Thermo Fisher) per the manufacturer instructions (AAV9 and AAV9s).
Large scale crude preps were treated with benzonase (250 U/mL, 1 h, 37° C.) before the centrifugation step (1 h, 10,000 rpm, 20° C.), then filtered using a 0.2 m Nalgene Rapid-Flow filter. Vectors were purified by affinity chromatography using either HiTrap columns prepacked with 1 mL AVB Sepharose HP (25-4112-11, GE Healthcare) (AAV3 and AAV3s) or a 5×125 mm Econoline column (TAC05/125PEO-AB-3, essentialLife Solutions) packed with 1 mL POROS CaptureSelect AAV9 affinity resin (Thermo Fisher) per the manufacturer instructions (AAV9 and AAV9s). Columns were sanitized with 5 column volumes (CV) 0.1 M H3PO4, 1 M NaCl, pH 2 (1 mL/min) and equilibrated with 5 CV PBS (21-030-CV, Corning) (1 mL/min). Clarified lysates were injected at 1 mL/min.
Columns were further washed with 10 CV PBS (1 mL/min). Vector particles were eluted in 3 mL 0.1 M NaOAc, 0.5 M NaCl, pH 2.5 (1 mL/min) and immediately neutralized with 400 μL of 1 M Tris-HCl, pH 10. Samples were further buffer-exchanged in PBS and concentrated by Amicon filtration (UFC910024, EMD Millipore) per the manufacturer instructions. Sample purity was assessed by SDS-PAGE, whereas DNAse I-resistant vector genomes were quantified by quantitative polymerase chain reaction (qPCR) using the TaqMan (Life Technologies) system with primers and probes targeting SV40 or eGFP.
All data were analyzed using R prior to normalization for reporting in the figures (unless otherwise indicated). P-values are reported in Table 2, below. Viral titers were compared using a paired, one-tailed Student'"'"'s t test and RNA levels were compared using a paired, two-tailed Student'"'"'s t test.
Table 2 shows the statistical analysis related to
AAP-HA: Complimentary oligonucleotides encoding the Hemagglutinin (HA) tag with BsiWI overhangs (5′ GTAC, 3′ CATG) were annealed in T4 Ligase Buffer ramping from 95° C. to 25° C. at 5°/min, PNK treated, and ligated into BsiWI digested and CIP treated AAV1 and AAV3 wt and AAPstop60 rep-cap plasmids.
CMV-HA-VP1 and CMV-VP3: gBlocks® Gene Fragments (IDT) of bp #4-696 of VP1 for AAV2, AAV3, Anc82, and 82DI were obtained, with the following modifications: an EcoRI site, start codon, and HA sequence added to 5′ end, ACG to ACC mutation of VP2 start codon, and ATG to CTG mutation of VP3 start codon. The gBlocks® include a BsrDI restriction site conserved in cap; gBlocks were digested with EcoRI and BsrDI. VP3 sequences were PCR amplified from the appropriate AAPstop60 rep-cap plasmids with primers incorporating 5′ EcoRI and 3′ HindIII restriction sites, then digested with either EcoRI and HindIII (for CMV-VP3) or BsrDI and HindIII (For CMV-HA-VP1). Fragments were ligated into pCDNA3.1(−) in the appropriate combinations. For CMV-AAP2, AAP was amplified from AAV2 rep-cap plasmid and ligated into pCDNA3.1(−).
Transfections were performed as in Crude Virus Preparation. At 36 h, supernatant was aspirated and cells lysed on plate with 100 μL (
For proteasome and lysosome inhibition experiments, media was removed 24 h after transfection and replaced with media containing the appropriate concentration of Bortezomib (Selleckchem PS-341), MLN7243 (Chemgood C-1233), Bafilomycin (Enzo BML-CM110-0100), or DMSO (for wt and AAPstop untreated samples) and incubated an additional 8 h. 25 μg total protein were loaded per well (diluted 1:10 for loading control). For protein turnover experiments by blocking protein synthesis, media was removed 24 h after transfection and replaced with media containing 50 μg/mL CHX (Sigma C7698) and lysates were harvested as described above at 1, 2, 4, 6, and 8 h time points, and for the 0 h time point media was not replaced but lysates were harvested. 10% FBS was maintained throughout all transfections and drug incubations described here.
Electrophoresed proteins were transferred to PVDF membranes, incubated with primary antibody (B1, 1:250, ARP#03-65158; Actin, 1:20000, Abcam 8227; Tubulin, 1:20000, Abcam 7291; HA, 1:5000, Abcam 9110; p62, 1:1000, Cell Signalling 5114) overnight, and detected with Anti-mouse (GE Healthcare LNXA931/AE) or Anti-rabbit (Sigma A0545) HRP conjugated secondary antibody and Thermo Super Signal® West Pico or Femto.
For dot blots, protein lysates were diluted 1:100 and 2 μL was spotted onto nitrocellulose membranes, allowed to dry, blocked in 5% milk, and incubated with ADK8 (ARP#03-651160) overnight.
PEI transfections were performed with 10 μg each of CMV-HA-VP1, CMV-VP3, and CMV-AAP2 plasmid of the appropriate serotype (CMV-AAP2 added only where indicated) on 10 cm dishes of HEK 293 cells at ˜80% confluency. PEI Max (Polysciences)/DNA ratio was maintained at 1.375:1 (w/w) in serum-free media. At 24 hours post transfection, cells were pelleted and resuspended in 1 mL lysis buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris, pH8, plus cOmplete Mini™ protease inhibitor). Immunoprecipitation was performed with rb Anti-HA antibody (Abcam 9110) and Pierce Protein A/G Plus Agarose beads. Precipitated proteins were eluted in 4× NuPAGE® LDS sample buffer+0.5% βME at 90° C. for 10 minutes. 10 μL (IP) or 30 μL (input) were loaded and electrophoresed on NuPAGE® 4-12% Bis-Tris gels and detected in Western Blotting above.
For detection of full virions (
PEI transfections were performed with 2 μg each of CMV-HA-VP1, CMV-VP3, and CMV-AAP2 plasmid of the appropriate serotype (CMV-AAP2 added only where indicated) on 6-well plates of HEK 293 cells at ˜80% confluency. PEI Max (Polysciences)/DNA ratio was maintained at 1.375:1 (w/w) in serum-free media. After 36 h, on-plate lysis was performed with M-PER™ (Thermo) buffer supplemented with cOmplete™ mini protease inhibitor. Lysate was divided into three and treated with 5 mM final concentration of disuccinimidyl glutarate (DSG, Thermo), disuccinimidyl suberate (DSS, Thermo), or an equal volume of DMSO as a mock treatment. Reactions were incubated on ice for 1 h, mixing periodically, then quenched with 1 M Tris. NuPAGE® LDS Sample Buffer+2-Mercaptoethanol were added and samples boiled for 10 minutes, loaded onto SDS-PAGE gel, and interrogated by Western Blot with B1 antibody.
Transfections were performed as described in Crude Virus Preparations/Titration. RNA was harvested after 36 h with Qiagen RNeasy Mini® kit according to the manufacturer'"'"'s instructions. TURBO DNA-free™ kit (Invitrogen) was used according to manufacturer'"'"'s instructions (Rigorous DNase treatment protocol) to eliminate contaminating DNA from samples. cDNA synthesis was performed with iScript™ kit (BioRad) using 250 ng total RNA from each sample. Reactions were then diluted 10-fold, and 5 μL of diluted cDNA used in each qPCR reaction, prepared with PowerUp™ SYBR™ Green Master Mix (Applied Biosystems) and intron, intron-spanning, or GAPDH primers to detect unspliced, spliced, or housekeeping gene products.
For negative staining on chromatography-purified AAV3 and AAV3stop, vector samples (5 μL or 50 μL drop) and a blank buffer control were adsorbed onto 200 mesh carbon and formvar coated nickel grids, rinsed, and stained with 2% aqueous uranyl acetate for 30 seconds then absorbed off on filter paper and air dried. All grids were imaged using a FEI Tecnai G2 Spirit transmission electron microscope (FEI, Hillsboro, Oreg.) at 100 kV accelerating voltage, interfaced with an AMT XR41 digital CCD camera (Advanced Microscopy Techniques, Woburn, Mass.) for digital TIFF file image acquisition. TEM imaging of AAV samples were assessed and digital images captured at 2k×2k pixel, 16-bit resolution.
The appropriate serotype and GC particles of AAV-CMV-EGFP-T2A-Luc was added to HEK-293 cells on a 96-well plate pre-infected with human adenovirus 5 (hAd5) 24 h prior at a multiplicity of infection of 20 particles/cell. The cells were imaged with an EVOS® FL Imaging System at 24 and 48 h, after which D-luciferin containing buffer was added and luminescence was measured using Synergy H1 microplate reader (BioTek; Winooski, Vt.).
To test whether AAP is required to assemble capsids from the full complement of VP proteins (i.e., VP1, 2, and 3), AAP expression was abolished from rep-cap trans plasmids by an early stop codon in the AAP reading frame, a silent mutation in VP (
Recombinant AAV vectors were produced from AAPstop60 and wildtype AAP (WT) plasmids, and titrated by qPCR quantifying DNase resistant particles (DRP). AAPstop60 vector titers reveal that when all three VP proteins are present, AAP is not strictly required to assemble the virion in several serotypes (
The advantage of DRP titration is the ability to quantify virus of any capsid serotype with the same vector genome absent of differential bias in measurement. However, DRP measures the amount of assembled particles that also underwent viral genome packaging, a process that occurs downstream of capsid assembly. Moreover, DRP does not assess for non-packaged, empty AAV virions. To directly assay capsid assembly and rule out the possibility that serotypes with low AAPstop60 titers were due to a packaging defect, an A20 capsid ELISA was performed, which recognizes a conformational epitope only present in assembled AAV2 capsids. A20 cross-reacts with AAV3, allowing us to assay assembly directly for an AAV that requires AAP (AAV2) and one that accomplishes assembly in the AAPstop60 context (AAV3). A20 ELISA data for both serotypes corroborated the DRP indirect measure of assembly (
To ensure that the observed range of AAP dependence for assembly was not due to variation in VP translation efficiencies imposed by alternate codon usage in the AAPstop60 mutants, VP protein levels produced by WT and AAPstop60 constructs were interrogated. The B1 monoclonal antibody detects a conserved linear epitope on VP proteins in denaturing conditions across all AAVs tested in this study except AAV4 and rh32.33, allowing nearly all serotypes to be assayed. No appreciable difference in VP2 or VP3 protein levels, and a slight decrease in VP1 levels, was observed for AAPstop60s that produce 10% or higher of their respective WT titers (
To examine whether the observed decreases in VP levels were due to a potential translational defect, AAP2 was co-expressed in trans with AAPstop60 (rescue;
To interrogate degradation as the mechanism for instability, AAV8 AAPstop60 transfected cells were treated with increasing concentrations of the proteasome inhibitor Bortezomib, the E1 inhibitor MLN7243, or the vacuolar specific H+ATPase inhibitor Bafilomycin (
In an attempt to examine the rate of AAV8 VP degradation, protein synthesis was blocked with Cycloheximide (CHX) and protein lysates were harvested at progressive time points (
Given the spectrum of AAP phenotypes observed across the major clades, 9 putative evolutionary intermediates (AncAAVs) to the major AAV serotypes also were tested in order to gain insight into what elements of VP structure either impose the observed requirement for AAP or impart an ability for some VPs to perform these functions independently (Zinn et al., 2015, Cell Rep., 12:1056-68). As with the natural serotypes, a broad range of requirement for AAP was observed for the AAPstop60 AncAAVs (
Although the AAPstop60 early stop codon is placed upstream of domains shown to be required for AAP2 function, it is downstream of a highly conserved region (residues 52-57) in AAP (
For the six AAVs whose AAPstop60 produces at least 10% of WT titer and for AAV4, recently demonstrated to assemble VP3-only capsids without AAP (Earley et al., 2017, J. Virol., 91:e01980-16), further upstream stop constructs (AAPstop20) were generated, placing the early stop codon at residue ˜23 in the AAP ORF (silent mutations in VP). Of these, AAV5, rh32.33, and AAV4 AAPstop20 produce virus, while AAV3, AAV9, Anc110, and Anc113 do not (
Taken together with
Taking into consideration VP stability, AAPstop60 titers, and AAPstop20 titers, for clarity, the AAP phenotypes were categorized as (i) AAP-independent, (ii) AAPC-independent, and (iii) AAP-dependent (
To examine whether serotypes with different AAP phenotypes'"'"' VPs are subject to the same mechanisms of degradation, degradation of AAV3 proteins were blocked in the same manner as previously performed for AAV8 (
While AAPN alone facilitates the production of appreciable quantities of virus for many serotypes, whether AAPN-assembled particles retain the proper morphology as well as infectivity functions was next addressed. TEM imaging of AAV3 WT and AAV3AAPstop60 vectors indicate identical gross particle morphologies (
As a next step toward identifying VP structure responsible for assembly functions, an overview of how AAP phenotypes diverge across the wide genetic range of AAV capsids tested was sought, aiming to identify phenotypic differences across small genetic distances. AAP phenotypes of the 12 natural serotypes and the nine ancestral variants were correlated to the reconstructed phylogeny (
The observation that AAP phenotypes have branch-specific trends within the phylogeny presented the opportunity for a facile method to identify elements of VP structure critical for assembly functions. Given that VP sequence diverges by small increments along each of these branches, it was hypothesized that a set of residues homologous only within the members of their respective branch were likely to functionally contribute to capsid assembly. To this end, a multiple sequence alignment was generated with the Branch D and Branch I members. Within the alignment, a total number of 149 positions varied, however, at only twelve positions, the residue is conserved within Branch I, with a different yet shared identity on Branch D. Of these twelve, eight individual residues and two pairs of adjacent residues comprise 10 sites on VP. At some of these sites, residue identity diverges within Branch I members; however, they share a chemical property that contrasts with Branch D. For example, site 1 is a basic lysine in Branch D serotypes, compared to a threonine in Branch I for Anc110 and rh8, and a serine in AAV9, both hydroxylic residues. The approach to identify a phenotypic switch along a reconstructed phylogeny and then interrogate the conserved differences across the two diverging lineages for the phenotype of interest in order to map the structural determinant(s) responsible was named phenotype-to-phylogeny mapping.
To test whether the 10 sites constitute a motif that functions in capsid assembly, the Branch I identities were engrafted onto a member of Branch D and tested to determine whether the resulting hybrid gains AAPC-independent assembly function. Anc82, the node from which Branch I diverges, was chosen as the background for these mutations; as the closest relative to the Branch I serotypes it is more likely to tolerate several targeted mutations and retain functionality than a more distant relative. All ten sites in Anc82 were mutated to Branch I identities en masse by site-directed mutagenesis, creating a variant named 82DI (
Whether this DI motif affects VP protein stability was next assessed. Consistent with other AAP-dependent serotypes (
To properly categorize 82DI'"'"'s AAP phenotype, AAPstop20 for Anc82 and DI were generated, and loss of protein in 82DIstop20 was observed, indicating that 82DI is AAPC-independent (
To assess the broader impact of AAPC-independent assembly on the capsid as a whole, 82DI'"'"'s biophysical properties and transduction capabilities was further characterized compared to its parental strain, Anc82. The Tm of 82DI is 5° C. lower than Anc82 (
To examine how this motif influences particle assembly, where these residues lie within the 3-dimensional fold of VP and within an assembled capsid were mapped. Although crystal structures of AncAAVs are not available, the terminal Branch D (AAV8) and Branch I serotypes (AAV9) have been solved (DiMattia et al., 2012, J. Virol., 86:6947-58; Nam et al., 2007, J. Virol., 81:12260-71), and were used as surrogates to map the DI motif. Two of the 10 sites lie in the unstructured region of the VP N-terminus, but only site 1 is outside of VP3. Of the eight sites within the structured region of VP, seven of them map to the three-fold interface of a VP trimer and contact a neighboring monomer (
Comparing an AAV8 (AAP-dependent) trimer to an AAV9 (AAPC-independent) trimer at the atomic level, most of these sites exhibit compelling evidence for stronger inter-monomeric interactions within an AAPC-independent trimer than in the AAP-dependent trimer (
On AAV9, a hydrogen bond forms between a conserved asparagine and a glutamine at site 8 of an adjacent VP monomer. On AAV8, this bond is unable to materialize due to a Gln to Ala substitution (
Next, it was hypothesized that VPs of AAPC-independent AAVs are able to associate into oligomers in the absence of AAPC, whereas AAP-dependent serotypes'"'"' VPs do not strongly associate unless a full-length AAP is present. To test this theory, VP-VP interaction of AAP-dependent and AAPC-independent AAVs were evaluated by co-immunoprecipitation of VP3 with HA-tagged VP1 as bait (
Addition of AAP2 allowed VP3 co-precipitation in AAP-dependent serotypes, and co-precipitated an unknown VP species between VP2 and VP3'"'"'s predicted molecular weights in the AAPC-independent serotypes (marked with *,
To begin examining whether AAP is promoting oligomerization into species of defined geometry such as trimers or pentamers, or whether the increase in VP-VP interactions observed by co-IP are more randomized associations, crosslinking agents were added to transfected cell lysates and VP species interrogated by Western Blot (
To further ensure that the VP oligomerization process was being interrogated separately from their assembly into full capsids, the IP experiment was repeated with AAV2 VPs, adding rep, aap2, helper, and ITR-flanked reporter genome plasmids in trans to allow quantification of assembled DRPs (
It is to be understood that, while the methods and compositions of matter have been described herein in conjunction with a number of different aspects, the foregoing description of the various aspects is intended to illustrate and not limit the scope of the methods and compositions of matter. Other aspects, advantages, and modifications are within the scope of the following claims.
The disclosed methods, compositions, and other materials are disclosed as described herein, but it is understood that combinations, subsets, interactions, groups, etc. of these methods, compositions, and other materials are also disclosed. That is, while specific reference to each various individual and collective combinations and permutations of these compositions and methods may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular composition of matter or a particular method is disclosed and discussed and a number of compositions or methods are discussed, each and every combination and permutation of the compositions and the methods are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed.