Methods for predicting ancestral viral sequences and uses thereof

Abstract

The present invention provides methods of predicting ancestral viral sequences and uses thereof. Methods for predicting ancestral sequences of viruses or portions thereof are described. Also described are predicted ancestral sequences of adeno-associated virus (AAV) capsid polypeptides. The present disclosure also provides methods of gene transfer and methods of vaccinating a subject by administering a target antigen operably linked to an AAV capsid polypeptide.

Description

[0001] This application is a divisional application of Chinese invention patent application filed on October 10, 2014, entitled "Method for predicting ancestral virus sequences and its use", PCT application number: PCT / US2014 / 060163, and patent application number: 201480065410.2. Technical Field

[0002] Generally speaking, this disclosure relates to viruses. Background Technology

[0003] Circumventing and avoiding neutralizing or toxic immune responses to gene therapy vectors is a major challenge for all types of gene transfer vectors. Gene transfer has been most effectively achieved to date using vectors based on viruses circulating in humans and animals (e.g., adenoviruses and adeno-associated viruses (AAVs)). However, if the subject is already naturally infected with the virus, subsequent treatment with that virus-based vector leads to increased safety risks and reduced efficiency due to cellular and humoral immune responses. Viral capsid antigens are primarily responsible for innate and / or adaptive immunity against viral particles; however, peptides encoded by viral genes can also be immunogenic. Summary of the Invention

[0004] This disclosure describes methods for predicting and synthesizing ancestral viral sequences or portions thereof, and also describes viral particles containing such ancestral viral sequences. The methods described herein are applied to adeno-associated viruses (AAVs); therefore, this disclosure describes predicted ancestral AAV sequences and AAV viral particles containing such ancestral AAV sequences. This disclosure also describes the reduced seroprevalence exhibited by viral particles containing ancestral sequences relative to viral particles containing contemporary sequences.

[0005] In one aspect, this disclosure includes adeno-associated virus (AAV) capsid polypeptides, such as synthetic and / or artificial AAV capsid polypeptides, having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17. In some embodiments, the AAV capsid polypeptide or viral particles containing the AAV capsid polypeptide exhibit a lower seropositivity rate than the AAV2 capsid polypeptide or viral particles containing the AAV2 capsid polypeptide, and the AAV capsid polypeptide or viral particles containing the AAV capsid polypeptide exhibit substantially the same or lower seropositivity rate as the AAV8 capsid polypeptide or viral particles containing the AAV8 capsid polypeptide. In some embodiments, the AAV capsid polypeptide or viral particles containing the AAV capsid polypeptide are neutralized by human serum to a lesser extent than the AAV2 capsid polypeptide or viral particles containing the AAV2 capsid polypeptide, and the AAV capsid polypeptide or viral particles containing the AAV capsid polypeptide are neutralized by human serum to a similar or lesser extent than the AAV8 capsid polypeptide or viral particles containing the AAV8 capsid polypeptide. In some embodiments, the AAV capsid polypeptide is purified. The AAV shell polypeptide provided in this article can be encoded by nucleic acid sequences selected from the group consisting of: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 and 18.

[0006] In one aspect, this disclosure provides nucleic acid molecules, such as synthetic and / or artificial nucleic acid molecules, encoding adeno-associated virus (AAV) capsid polypeptides having nucleic acid sequences selected from the group consisting of: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, and 18. Vectors comprising such nucleic acids and host cells comprising such vectors are also provided.

[0007] In another aspect, this disclosure provides purified viral particles that include the AAV capsid polypeptide described herein. In some embodiments, the viral particles contain transgenes.

[0008] In other respects, this disclosure provides adeno-associated virus (AAV) capsid polypeptides, such as synthetic and / or artificial AAV capsid polypeptides, having at least 95% (e.g., 97, 98, 99, or 100%) sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, and 26. In some embodiments, the AAV capsid polypeptide or viral particles containing the AAV capsid polypeptide exhibit a lower seropositivity rate than the AAV2 capsid polypeptide or viral particles containing the AAV2 capsid polypeptide, and the AAV capsid polypeptide or viral particles containing the AAV capsid polypeptide exhibit substantially the same or lower seropositivity rate than the AAV8 capsid polypeptide or viral particles containing the AAV8 capsid polypeptide. In some embodiments, the AAV capsid peptide or viral particles containing the AAV capsid peptide are neutralized by human serum to a lesser extent than the AAV2 capsid peptide or viral particles containing the AAV2 capsid peptide, and the AAV capsid peptide or viral particles containing the AAV capsid peptide are neutralized by human serum to a similar or lesser extent than the AAV8 capsid peptide or viral particles containing the AAV8 capsid peptide. In some embodiments, the AAV capsid peptide is purified.

[0009] In another aspect, the AAV capsid polypeptide described herein can be encoded by a nucleic acid sequence as described herein. In one embodiment, this disclosure provides a nucleic acid molecule encoding an adeno-associated virus (AAV) capsid polypeptide, wherein said nucleic acid molecule has at least 95% (e.g., 97, 98, 99, or 100%) sequence identity with a nucleic acid as described herein. This disclosure also provides vectors comprising such nucleic acid molecules, and host cells comprising such vectors.

[0010] In another aspect, this disclosure provides viral particles comprising at least one of the AAV capsid polypeptides described herein. In some embodiments, the viral particles include transgenes.

[0011] In some aspects, this disclosure provides methods for administering viral particles as described herein to subjects requiring gene transfer or vaccination. In some embodiments, the viral particles exhibit a lower seropositivity rate than AAV2 viral particles. In some embodiments, the viral particles exhibit substantially the same or lower seropositivity rate as AAV8 viral particles. In some embodiments, the viral particles are neutralized by human serum to a lesser extent than AAV2 viral particles, and the AAV viral particles are neutralized by human serum to a similar or lesser extent than AAV8 viral particles.

[0012] In one aspect, this disclosure provides a method for administering a target antigen operatively linked to an AAV capsid peptide as described herein to a subject in need of vaccination. In some embodiments, the AAV capsid peptide exhibits a lower seropositivity rate than the AAV2 capsid peptide. In some embodiments, the AAV capsid peptide exhibits substantially the same or lower seropositivity rate than the AAV8 capsid peptide. In some embodiments, the AAV capsid peptide is neutralized by human serum to a lesser extent than the AAV2 capsid peptide, and is neutralized by human serum to a similar or lesser extent than the AAV8 capsid peptide.

[0013] In another aspect, this disclosure provides a computer method (insilico method) for predicting the sequence of an ancestral virus or a portion thereof. Such a method typically involves providing nucleotide or amino acid sequences from multiple contemporary viruses or portions thereof; aligning the sequences using a multiple sequence alignment (MSA) algorithm; performing evolutionary modeling to obtain predicted ancestral phylogeny for the multiple contemporary viruses or portions thereof; estimating the evolutionary probability of a specific nucleotide or amino acid residue at each position in the sequence at the phylogenic node of the predicted ancestral phylogeny; and predicting the sequence of the ancestral virus or a portion thereof based on the estimated probability at each position.

[0014] In some embodiments, a computer processor is used to perform one or more of the steps. In some embodiments, the MSA algorithm uses phylogenetic information to predict whether a gap in the alignment is the result of a deletion or insertion. In some embodiments, the MSA algorithm is a Probabilistic Alignment Kit (PRANK). In some embodiments, the Aikake Information Criterion (AIC) is used to select a model for evolutionary modeling. In some embodiments, the predicted ancestral phylogenetics are obtained using the JTT model and the gamma distribution model ("+G") and the frequency calculation of πi ("+F"). In some embodiments, the evolutionary modeling step is performed using the JTT+G+F model. In some embodiments, the method includes synthesizing an ancestral virus or a portion thereof based on the predicted sequence. In some embodiments, the method includes assembling an ancestral virus or a portion thereof into ancestral virus particles.

[0015] In some embodiments, the method further includes screening ancestral virus particles for at least one of the following: (b) replication; (c) gene transfer properties; (d) receptor binding; or (e) seropositivity. In some embodiments, the ancestral virus particles exhibit a lower seropositivity than virus particles assembled from at least one of a plurality of contemporary viruses or portions thereof. In some embodiments, the ancestral virus particles are neutralized by human serum to a lesser extent than virus particles assembled from at least one of a plurality of contemporary viruses or portions thereof. In some embodiments, the plurality of contemporary viruses or portions thereof belong to a family selected from the group consisting of: adenovirus (AV), human immunodeficiency virus (HIV), retrovirus, lentivirus, herpes simplex virus (HSV), vaccinia virus, pox virus, influenza virus, respiratory syncytial virus, parainfluenza virus, and foamy virus.

[0016] Therefore, this disclosure provides ancestral viruses or portions thereof that exhibit reduced susceptibility to pre-existing immunity in the current population compared to contemporary viruses or portions thereof. Typically, the reduced susceptibility to pre-existing immunity exhibited by ancestral viruses or portions thereof in the current population reflects reduced susceptibility to neutralizing antibodies.

[0017] The present invention provides the following content.

[0018] 1. An adeno-associated virus (AAV) capsid polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 and 17.

[0019] 2. The AAV capsid peptide of embodiment 1, wherein the AAV capsid peptide or viral particles containing the AAV capsid peptide exhibit a lower seroprevalence than the AAV2 capsid peptide or viral particles containing the AAV2 capsid peptide, and wherein the AAV capsid peptide or viral particles containing the AAV capsid peptide exhibit the same or lower seroprevalence than the AAV8 capsid peptide or viral particles containing the AAV8 capsid peptide.

[0020] 3. The AAV capsid polypeptide of embodiment 1, wherein the AAV capsid polypeptide or viral particles containing the AAV capsid polypeptide are neutralized by human serum to a lesser extent than the AAV2 capsid polypeptide or viral particles containing the AAV2 capsid polypeptide, and wherein the AAV capsid polypeptide or viral particles containing the AAV capsid polypeptide are neutralized by human serum to a similar or lesser extent than the AAV8 capsid polypeptide or viral particles containing the AAV8 capsid polypeptide.

[0021] 4. The AAV shell polypeptide of any one of embodiments 1-3, wherein the AAV shell polypeptide is purified.

[0022] 5. The AAV shell polypeptide of embodiment 1, encoded by nucleic acid sequences selected from the group consisting of: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 and 18.

[0023] 6. A nucleic acid molecule encoding an adeno-associated virus (AAV) capsid polypeptide having a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 and 18.

[0024] 7. A vector containing the nucleic acid molecule of implementation scheme 6.

[0025] 8. A host cell containing the vector of implementation scheme 7.

[0026] 9. Purified viral particles containing the AAV capsid polypeptide of any one of embodiments 1-5.

[0027] 10. The purified virus particles of embodiment 9, which further contain genetically modified organisms.

[0028] 11. An adeno-associated virus (AAV) capsid polypeptide having at least 95% sequence identity with an amino acid sequence selected from the group consisting of: SEQ ID NO: 19, 20, 21, 22, 23, 24, 25 and 26.

[0029] 12. The AAV capsid peptide of embodiment 11, wherein the AAV capsid peptide or viral particles containing the AAV capsid peptide exhibit a lower seropositivity rate than the AAV2 capsid peptide or viral particles containing the AAV2 capsid peptide, and wherein the AAV capsid peptide or viral particles containing the AAV capsid peptide exhibit the same or lower seropositivity rate than the AAV8 capsid peptide or viral particles containing the AAV8 capsid peptide.

[0030] 13. The AAV capsid polypeptide of embodiment 11, wherein the AAV capsid polypeptide or viral particles containing the AAV capsid polypeptide are neutralized by human serum to a lesser extent than the AAV2 capsid polypeptide or viral particles containing the AAV2 capsid polypeptide, and wherein the AAV capsid polypeptide or viral particles containing the AAV capsid polypeptide are neutralized by human serum to a similar or lesser extent than the AAV8 capsid polypeptide or viral particles containing the AAV8 capsid polypeptide.

[0031] 14. The AAV shell polypeptide of any one of embodiments 11-13, wherein the AAV shell polypeptide is purified.

[0032] 15. An AAV shell polypeptide of any one of embodiments 11-14, wherein the polypeptide has at least 99% sequence identity with an amino acid sequence selected from the group consisting of: SEQ ID NO: 19, 20, 21, 22, 23, 24, 25 and 26.

[0033] 16. An AAV shell polypeptide of any one of embodiments 11-14, wherein the polypeptide has 100% sequence identity with an amino acid sequence selected from the group consisting of: SEQ ID NO: 19, 20, 21, 22, 23, 24, 25 and 26.

[0034] 17. Viral particles comprising at least one of the AAV capsid polypeptides of any one of embodiments 11-16.

[0035] 18. The viral particle of implementation scheme 17, which further contains genetically modified organisms.

[0036] 19. A method for gene transfer or vaccination using transgenic technology, said method comprising:

[0037] The viral particles of Implementation Scheme 10 or Implementation Scheme 18 are administered to subjects requiring gene transfer or immunization, wherein the viral particles exhibit a lower seropositivity rate than AAV2 viral particles.

[0038] 20. The method of embodiment 19, wherein the viral particles exhibit substantially the same or lower seropositivity rate as AAV8 viral particles.

[0039] 21. The method of embodiment 19, wherein the virus particles are neutralized by human serum to a lesser extent than AAV2 virus particles, and wherein the AAV virus particles are neutralized by human serum to a similar or lesser extent than AAV8 virus particles.

[0040] 22. A method for administering a vaccine to a subject, the method comprising:

[0041] The target antigen of the AAV shell peptide operably linked to Embodiment 1 or Embodiment 11 is administered to subjects requiring vaccination, wherein the AAV shell peptide exhibits a lower seropositivity rate than the AAV2 shell peptide.

[0042] 23. The method of embodiment 22, wherein the AAV shell peptide exhibits approximately the same or lower seropositivity rate as the AAV8 shell peptide.

[0043] 24. The method of embodiment 22, wherein the AAV shell polypeptide is neutralized by human serum to a lesser extent than the AAV2 shell polypeptide, and the AAV shell polypeptide is neutralized by human serum to a similar or lesser extent than the AAV8 shell polypeptide.

[0044] 25. A computer method (in silico method) for predicting the sequence of an ancestral virus or a portion thereof, said method comprising:

[0045] Provide nucleotide or amino acid sequences from multiple contemporary viruses or portions thereof;

[0046] The sequences were compared using the Multiple Sequence Alignment (MSA) algorithm;

[0047] Evolutionary modeling is used to obtain predicted ancestral phylogenetic relationships for the multiple contemporary viruses or parts thereof;

[0048] At the phylogenetic node of the predicted ancestral phylogeny, the evolutionary probability of a specific nucleotide or amino acid residue at each position in the sequence is estimated, and

[0049] Predict the sequence of the ancestral virus or a portion thereof based on the estimated probability at each position.

[0050] 26. The method of implementation 25, wherein all steps are performed using a computer processor.

[0051] 27. The method of embodiment 25 or embodiment 26, wherein the MSA algorithm uses phylogenetic information to predict whether a gap in the alignment is the result of a missing or inserted object.

[0052] 28. The method of implementation 27, wherein the MSA algorithm is a Probabilistic Alignment Kit (PRANK).

[0053] 29. The method of any one of Implementation Schemes 25 to 28, wherein the Aikake Information Criterion (AIC) is used to select the model for evolutionary modeling.

[0054] 30. The method of any one of embodiments 25 to 28, wherein the predicted ancestral phylogeny is obtained using the JTT model and the gamma distribution model (“+G”) and the frequency calculation of πi (“+F”).

[0055] 31. The method of implementation scheme 25, wherein the step of using the JTT+G+F model for evolutionary modeling.

[0056] 32. The method of any one of embodiments 25 to 31, further comprising synthesizing the ancestral virus or a portion thereof based on the predicted sequence.

[0057] 33. The method of embodiment 32, further comprising assembling the ancestral virus or a portion thereof into ancestral virus particles.

[0058] 34. The method of implementation scheme 33, further comprising screening the ancestral viral particles for at least one of the following: (1) replication; (b) gene transfer properties; (c) receptor binding; or (d) seropositivity.

[0059] 35. The method of embodiment 34, wherein the ancestral viral particles exhibit a lower seropositivity rate than viral particles assembled from at least one of the plurality of contemporary viruses or portions thereof.

[0060] 36. The method of embodiment 34, wherein the ancestral virus particles are neutralized by human serum to a lesser extent than virus particles assembled from at least one of the plurality of contemporary viruses or portions thereof.

[0061] 37. The method of embodiment 25, wherein the plurality of contemporary viruses or portions thereof belong to a family selected from the group consisting of: adenovirus (AV), human immunodeficiency virus (HIV), retrovirus, lentivirus, herpes simplex virus (HSV), vaccinia virus, pox virus, influenza virus, respiratory syncytial virus, parainfluenza virus, and foamy virus.

[0062] 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 material compositions pertain. While methods and materials similar to or equivalent to those described herein may be used in the practice or testing of methods and material compositions, suitable methods and materials are described below. Furthermore, materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Attached Figure Description

[0063] This patent or application document contains at least one drawing made in color. A copy of the published text of this patent or application with the color drawing is provided to the authorities upon request and payment of the necessary fees.

[0064] Figure 1 This is a schematic diagram illustrating the relationship between ancestral virus / current virus infection and ancestral host / current host immune response.

[0065] Figure 2 a) through d) are a series of schematic diagrams illustrating instances of the ancestral reconstruction process. The data shown are extracted from the complete dataset and represent residues 564-584 (AAV2-VP1 numbers).

[0066] Figure 3 The phylogenetic tree of contemporary AAV sequences generated using the method described in this paper is shown.

[0067] Figure 4 The alignment of the ancestral AAV VP1 peptide is shown.

[0068] Figure 5A and 5B Together, they show a comparison between the functional ancestral AAV VP1 peptide and the contemporary AAV VP1 peptide.

[0069] Figure 6 The image is a gel electrophoresis diagram that demonstrates that the ancestral AAV VP1 sequence was transcribed and alternatively spliced ​​in a manner similar to that of the contemporary AAV VP1 sequence.

[0070] Figure 7 This is a graph showing the luciferase activity in HEK293 cells transduced with the ancestral AAV vector.

[0071] Figure 8 This is a graph showing that ancestral AAV vectors are more resistant to IVIG neutralization than contemporary AAV vectors.

[0072] Figure 9This is a graph showing the sequence comparison between the Anc80 library and Anc80L65 (from diagonal upwards, %, with amino acid differences below #).

[0073] Figure 10 The images in Figure A and Figure B are images of the experimental results, demonstrating that Anc80L65 can assemble and produce high-titer particles. Figure A shows that Anc80L65 can produce vector yields comparable to AAV2; Figure B is a TEM image of viral particles including Anc80L65; Figure C shows viral particles including Anc80L65, based on SDS-PAGE gel under denaturing conditions, which are able to produce AAV cap VP1, 2, and 3 proteins; and Figure D shows a Western blot of Anc80L65 using AAV capsid antibody B1.

[0074] Figure 11 Figures A and C are images of the experimental results, demonstrating that using GFP as a readout (Figure A) or luciferase (Figure B), Anc80L65, relative to AAV2 and / or AAV8 controls, can effectively infect HEK293 cells in vitro and effectively target the liver after IV injection of AAV encoding the nuclear LacZ transgene (top row, Figure C: liver), direct IM injection of AAV encoding GFP (middle row, Figure C: muscle), and subretinal injection of AAV encoding GFP (bottom row, Figure C: retina).

[0075] Figure 12 The AD figures show that Anc80L64 has a minimal seropositivity rate in human populations using IVIg (Figure A) or serum from the Belgian population (Figure B), serum from the Boston population (Figure C), or serum from Cynomolgus macaques (Figure D).

[0076] Figure 13A and 13B This is a sequence identity matrix generated using MAFFT, showing the amino acid sequence of the VP1 protein from the ancestral vector compared to the amino acid sequence of the VP1 protein of representative existing AAVs. Figure 13A ), and the amino acid sequence of the VP3 protein of the ancestral vector compared with the amino acid sequence of the VP3 protein of a representative extant AAV (). Figure 13B ).

[0077] Figure 14 This graph shows the production of AAV vectors in triplicate on a small scale (6-well plates). The crude virus was evaluated by qPCR to determine the absolute production of each vector.

[0078] Figure 15It is a table showing the titer of each carrier, which is averaged and compared with the titer of AAV8.

[0079] Figure 16 The image shows the experimental results of adding 1.9E3 GC / cell for each vector to HEK293 cells (an MOI of 2.5E2–3.1E2 GC / cell was achieved in the case of Anc126, except for Anc126). Infectivity was assessed by fluorescence microscopy after 60 hours.

[0080] Figure 17 This is a graph showing the experimental results, where the cleavage originates from... Figure 16 The same cells were used, and luciferase expression was measured. (Compared to...) Figure 16 Similar to the previous example, Anc126 did not use other vectors to control the titer, but the range was 2.5E2 - 3.1E2 GC / cell MOI.

[0081] Figure 18 The table shows the luminescence of cells transduced by each vector, which is averaged and compared with the luminescence of AAV8.

[0082] Figure 19 The figure provides a summary of in vitro experiments determining the relative production and infectivity of the ancestral AAV vectors described in this paper. Detailed Implementation

[0083] Gene transfer (for experimental or therapeutic purposes) relies on vectors or vector systems to shuttle genetic information into target cells. Vectors or vector systems are considered major determinants of the efficiency, specificity, host response, pharmacology, and lifespan of gene transfer responses. Currently, the most efficient and effective way to achieve gene transfer is through the use of vectors or vector systems based on viruses that have been modified to be replication-defective.

[0084] However, seropositivity studies indicate that a significant proportion of the global human population has been pre-exposed (e.g., through natural infection) to a large number of viruses currently used in gene transfer and therefore possesses pre-existing immunity. It is known that neutralizing antibodies against viral vectors in these pre-exposed individuals sometimes significantly limit the extent of gene transfer or even redirect the virus away from its target. See, for example, Calcedo et al., (2009, J. Infect. Dis., 199:381-90) and Boutin et al., (2010, Human Gene Ther., 21:704-12). Therefore, this disclosure is based on the understanding that ancestral viruses or portions thereof exhibit reduced susceptibility to pre-existing immunity (e.g., reduced susceptibility to neutralizing antibodies) in the current human population compared to contemporary viruses or portions thereof.

[0085] Figure 1 This is a schematic diagram illustrating the relationship between ancestral and contemporary viral infections and ancestral and contemporary host immune responses. Figure 1 This demonstrates how ancestral AAVs could refractory to contemporary pre-existing immunity. It is hypothesized that contemporary extant viruses (Vc) evolved primarily from ancestral species (Vanc) through immune escape mechanisms under evolutionary pressure from host immunity. Each of these species, Vanc and Vc, possesses the ability to induce adaptive immunity, including B-cell and T-cell immunity (Ianc and Ic, respectively). It is hypothesized, and confirmed in this paper, that immunity induced by contemporary viruses does not necessarily cross-react with ancestral viral species, which may be substantially different from extant viruses in terms of epitope composition.

[0086] This disclosure provides a method for predicting sequences of ancestral viruses or portions thereof. One or more ancestral virus sequences predicted using the methods described herein can be generated and assembled into viral particles. As demonstrated herein, viral particles assembled from predicted ancestral virus sequences can exhibit smaller, and sometimes significantly smaller, seropositivity rates than current contemporary viral particles. Therefore, the ancestral virus sequences disclosed herein are suitable for use in vectors or vector systems for gene transfer.

[0087] Methods for predicting and synthesizing ancestral viral sequences

[0088] To predict ancestral viral sequences, nucleotide or amino acid sequences are first compiled from multiple contemporary viruses or portions thereof. Although the methods described herein use adeno-associated virus (AAV) capsid sequences as an example, the same methods can be applied to other sequences from AAV (e.g., the entire genome, rep sequences, ITR sequences) or any other virus or portions thereof. Viruses other than AAV include, but are not limited to, adenovirus (AV), human immunodeficiency virus (HIV), retroviruses, lentiviruses, herpes simplex virus (HSV), measles virus, vaccinia virus, poxvirus, influenza virus, respiratory syncytial virus, parainfluenza virus, foamy virus, or any other virus presumed to be immunologically problematic.

[0089] Sequences from as few as two contemporary viruses or their parts can be used; however, it should be understood that a larger number of sequences from contemporary viruses or their parts are desirable in order to include as much of the landscape as possible of modern sequence diversity, and also because a larger number of sequences can increase the predictive power of the algorithms described and used. For example, sequences from 10 or more contemporary viruses or their parts can be used, sequences from 50 or more contemporary viruses or their parts can be used, and sequences from 100 or more contemporary viruses or their parts can be used.

[0090] Such sequences can be obtained, for example, from many public databases, including but not limited to GenBank, UniProt, EMBL, the International Nucleotide Sequence Database Collaboration (INSDC), or the European Nucleotide Archive. Alternatively, such sequences can be obtained from organism-specific databases (e.g., HIV databases). Contemporary sequences may correspond to the entire genome, or may use only a portion of the genome, such as, but not limited to, sequences encoding one or more components of the viral capsid, replication proteins, or ITR sequence.

[0091] Next, the current generation of sequences is compared using the multiple sequence alignment (MSA) algorithm. Figure 2 (a) is a schematic diagram showing the alignment of multiple sequences. The MSA algorithm is well known in the field and is generally designed to work with datasets of different sizes and different inputs (e.g., nucleic acids or proteins), as well as to align sequences in a specific way (e.g., dynamic programming, progressive, heuristic) and apply different scoring schemes in the alignment (e.g., matrix-based or consistency-based, e.g., minimum entropy, pair summation, similarity matrix, gap score). Well-known MSA algorithms include, for example, ClustalW (Thompson et al., 1994, Nuc. Acids Res., 22:4673-90), Kalign (Lassmann et al., 2006, Nuc. Acids Res., 34:W596-99), MAFFT (Katoh et al., 2005, Nuc. Acids Res., 33:511-8), MUSCLE (Edgar, 2004, BMC Bioinform., 5:113), and T-Coffee (Notredame et al., 2000, J. Mol. Biol., 302:205-17).

[0092] As described herein, one of the key features when choosing the MSA algorithm for use in the methods described herein is how the algorithm handles gaps in alignments. A penalty value can be assigned to gaps in sequence alignments, depending on or independent of the gap size. Preferably, in contrast to biased, non-systematic treatment of gaps caused by, for example, insertions and / or deletions, the MSA algorithm used in the methods described herein applies phylogenetic information to predict whether a gap in an alignment is a result of deletion or insertion. Suitable methods for handling gaps in alignment and evolutionary analysis are described in Loytynoja and Goldman, 2008, Science, 320:1632-5, and commercially available algorithms for applying gaps to alignments in a manner suitable for use in the methods described herein are the Probabilistic Alignment Kit (PRANK; Goldman Group Software; Loytynoja and Goldman, 2005, PNAS USA, 102:10557-62) and variations of the PRANK algorithm.

[0093] Then, the evolutionary model is applied to the obtained alignments to obtain the predicted ancestral phylogeny (see [link]). Figure 2 (b)). Many evolutionary models are available in the art, each applying a slightly different matrix of substitution rates to amino acids. Algorithms applying these evolutionary models include, but are not limited to, the Dayhoff model (e.g., PAM120, PAM160, PAM250; Dayhoff et al., 1978, In Atlas of Protein Sequence and Structure (ed. Dayhoff), pp.345-52, National Biomedical Research Foundation, Washington DC), the JTT model (Jones et al., 1992, Comp. Appl. Biosci., 8:275-82), the WAG model (Whelan and Goldman, 2001, Mol. Biol. Evol., 18:691-9), and the Blosum model (e.g., Blosum45, Blosum62, Blosum80; Henikoff and Henikoff, 1992, PNAS USA, 89:10915-9).

[0094] Furthermore, for example, by considering that some positions are invariant (“+ I”; Reeves, 1992, J. Mol. Evol., 35:17-31), some positions undergo change at different rates (“+ G”; Yang, 1993, Mol. Biol. Evol., 10:1396-1401), and / or the equilibrium frequencies of nucleotides or amino acids are the same as those in the alignment (“+ F”; Cao et al., 1994, J. Mol. Evol., 39:519-27), the constraints imposed on evolutionary models by structure and function can be modeled themselves.

[0095] The fitness of one or more evolutionary models can be assessed using the Aikake Information Criterion (AIC; Akaike, 1973, In Second International Symposium on Information Theory, Petrov and Csaki, eds., pp 267-81, Budapest, Akademiai Kiado), the Bayesian Information Criterion (BIC; Schwarz, 1978, Ann. Statist. 6:461-4), or variations or combinations thereof. Furthermore, the AIC, BIC, or variations or combinations thereof can be used to assess the relative importance of including one or more parameters (e.g., the constraints discussed above) in an evolutionary model.

[0096] As explained in the Examples section below, based on the minimum AIC, ProTest3 (Darriba et al., 2011, Bioinformatics, 27(8):1164-5) can be used to determine that the JTT+G+F algorithm is the most suitable model for AAV evolution. Those skilled in the art will understand that the JTT+G+F algorithm can also be used to predict ancestral viral sequences different from the AAV capsid peptide; however, they will also understand that different evolutionary models may be more suitable depending on the dataset and fitness score.

[0097] Once an evolutionary model has been selected and its fitness determined, a phylogenetic tree of the viral sequence or a portion thereof can be constructed. Phylogenetic tree construction is known in the art and is typically performed using the maximum likelihood method, such as those performed by PhyML (Guindon and Gascuel, 2003, Systematic Biology, 52:696-704), MOLPHY (Adachi and Hasegawa, 1996, ed. Tokyo Institute of Statistical Mathematics), BioNJ (Gascuel, 1997, Mol. Biol. Evol., 14:685-95), or PHYLIP (Felsenstein, 1973, Systematic Biology, 22:240-9). Those skilled in the art will understand that a balance between computational complexity and goodness of fit is desirable in models involving amino acid substitutions.

[0098] The significance of a phylogenetic tree can be assessed as needed. Many statistical methods are available and routinely used to evaluate model significance, including but not limited to bootstrap, jackknife, cross-validation, permutation tests, or combinations or variations thereof. Significance can also be evaluated using, for example, the approximate likelihood-ratio test (aLRT; Anisimova and Gascuel, 2006, Systematic Biology, 55:539-52).

[0099] At any phylogenetic node in phylogeny (e.g., an internal phylogenetic node), a sequence can be reconstructed by estimating the evolutionary probability of a specific nucleotide or amino acid residue at each position in the sequence (e.g., Figure 2(c)). A phylogenetic node is an intermediate evolutionary branch point within a predicted ancestral phylogeny. As used herein, “evolutionary probability” refers to the probability of the presence of a specific nucleotide or amino acid at a particular position based on an evolutionary model, in contrast to a model that does not consider, for example, evolutionary shifts in codon selection. Exemplary models that take into account the evolutionary probability of a specific nucleotide or amino acid residue at a particular position can be evaluated using, for example, any number of maximum likelihood methods, including but not limited to phylogenetic analysis by maximum likelihood (PAML; Yang, 1997, Comp. Applic. BioSci., 13:555-6) or phylogenetic analysis using parsimony (PAUP; Sinauer Assoc., Inc., Sunderland, MA).

[0100] Based on the estimated evolutionary probability of specific nucleotide or amino acid residues at each position, predicted sequences of ancestral viruses or parts thereof can be assembled to form complete or partial synthetic nucleic acid or polypeptide sequences. As needed, the probability of any residue being in a given state at a given node can be calculated, and any position along the sequence with a calculated posterior probability below a specific threshold can be identified. Figure 2 (d) In this way, ancestral scaffold sequences can be generated, which may include variations at those locations with a probability below a certain threshold.

[0101] If the ancestral sequence predicted using the methods described herein is a nucleic acid sequence, then the sequence can be codon-optimized so that it can be efficiently translated into an amino acid sequence. Codon selection tables for different organisms are known in the art. Optionally, however, the codon selection table can be designed based on one or more contemporary sequences that have identity with the ancestral scaffold sequence (e.g., at least 90% sequence identity), and the ancestral sequence as described herein can be codon-optimized for mammalian (e.g., human) codon selection.

[0102] Any or all of the steps outlined in this article for predicting ancestral viral sequences can be performed or simulated on a computer (e.g., using a computer in silico) using a processor or microprocessor.

[0103] Ancestor Adeno-Associated Virus (AAV) Scaffold Sequence

[0104] The methods described herein are applied to adeno-associated virus (AAV) using contemporary capsid sequences (described in detail in the examples below). AAV is widely considered a therapeutic gene transfer vector and genetic vaccine vector, but exhibits a high seropositivity rate in the human population. Using the methods described herein, contemporary AAV sequences (see...) are employed. Figure 3 A phylogenetic tree was assembled, and predicted ancestral scaffold sequences were obtained at the specified phylogenetic nodes (Table 1). As used herein, an ancestral scaffold sequence refers to a sequence constructed using the methods described herein (e.g., using evolutionary probabilities and evolutionary modeling) and known to be non-existent in nature. As used herein, ancestral scaffold sequences differ from common sequences, which are typically constructed using the frequencies of nucleotide or amino acid residues at specific positions.

[0105] Table 1

[0106]

[0107] The scaffold sequence of the Anc80 polypeptide is shown in SEQ ID NO: 1, which is encoded by the scaffold sequence of the Anc80 nucleic acid shown in SEQ ID NO: 2. The Anc80 scaffold sequence contains 11 positions, at which either of two residues is possible. Therefore, the Anc80 scaffold sequence represents 2048 (2 11 ( ) different sequences.

[0108] To demonstrate the effectiveness of the methods described herein for predicting ancestral sequences of viruses or parts thereof, a library of 2048 predicted ancestral sequences at the AAV Anc80 node was generated, and as described herein, live viral particles were demonstrated to exhibit a lower seropositivity rate than viral particles assembled with contemporary capsid peptides, and in some cases, a significantly lower seropositivity rate.

[0109] Methods for preparing ancestral virus particles

[0110] After a predicted ancestral sequence of a virus or a portion thereof has been obtained, actual nucleic acid molecules and / or peptides can be produced (e.g., synthesized). Methods for producing artificial nucleic acid molecules or peptides based on sequences obtained, for example, by computer, are known in the art and include, for example, chemical synthesis or recombinant cloning. Other methods for producing nucleic acid molecules or peptides are known in the art and are discussed in more detail below.

[0111] Once the ancestral polypeptide has been generated, or once the ancestral nucleic acid molecule has been generated and expressed to produce the ancestral polypeptide, it can be assembled into ancestral viral particles using, for example, packaging host cells. Components of the viral particle (e.g., rep sequences, cap sequences, terminal inverted repeat (ITR) sequences) can be transiently or stably introduced into the packaging host cell using one or more vectors as described herein. One or more components of the viral particle can be based on predicted ancestral sequences as described herein, while the remaining components can be based on contemporary sequences. In some cases, the entire viral particle can be based on a predicted ancestral sequence.

[0112] These ancestral viral particles can be purified using conventional methods. As used herein, "purified" viral particles refer to viral particles taken from components of the mixtures in which they are prepared, such as, but not limited to, viral components (e.g., rep sequences, cap sequences), packaged host cells, and partially or incompletely assembled viral particles.

[0113] Once assembled, ancestral viral particles can be screened for, for example, replication capacity; gene transfer properties; receptor binding capacity; and / or seropositivity in a population (e.g., a human population). Determining whether a viral particle can replicate is routine in the art and typically involves infecting a host cell with a given amount of viral particles and determining whether the number of viral particles increases over time. Determining whether a viral particle is capable of performing gene transfer is also routine in the art and typically involves infecting a host cell with viral particles containing a transgene (e.g., a detectable transgene, such as a reporter gene, discussed in more detail below). Following viral infection and clearance, the presence or absence of the transgene in the host cell can be assessed. Determining whether a viral particle binds to its receptor is routine in the art, and such methods can be performed in vitro or in vivo.

[0114] Determining the seropositivity of viral particles is routinely performed in the art and typically involves using immunoassays to determine the prevalence of one or more antibodies in a sample (e.g., a blood sample) from a specific population of individuals. Seropositivity is understood in the art to refer to the proportion of subjects in a population who are seropositive (i.e., have been exposed to a specific pathogen or immunogen) and is calculated as the number of subjects in the population who have produced antibodies against a specific pathogen or immunogen divided by the total number of individuals in the population examined. Immunoassays are well known in the art and include, but are not limited to, immunodot, Western blot, enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA). As noted herein, ancestral viral particles exhibit a lower seropositivity rate than contemporary viral particles (i.e., viral particles assembled using contemporary viral sequences or portions thereof). For example, see Xuet 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); and Sakhria et al. (2013, PLoS Negl. Trop. Dis., 7:e2429), each of which determines the seropositivity rate of a specific antibody in a given population.

[0115] As described in this article, ancestral viral particles are neutralized by an individual's (e.g., a patient's) immune system to a lesser extent than contemporary viral particles. Several methods are available for determining the level of neutralizing antibodies in serum samples. For example, a neutralizing antibody assay measures the titer of antibody concentrations in experimental samples that neutralize infection by 50% or more compared to control samples without antibodies. See also Fisher et al. (1997, Nature Med., 3:306-12) and Manning et al. (1998, Human Gene Ther., 9:477-85).

[0116] With regard to the ancestral AAV capsid peptide exemplified herein, seropositivity and / or the degree of neutralization can be compared, for example, with the AAV8 capsid peptide or viral particles comprising the AAV8 capsid peptide, or the AAV2 capsid peptide or viral particles comprising the AAV2 capsid peptide. It is generally understood in the art that the AAV8 capsid peptide or viral particles exhibit a perceived lower seropositivity and resulting neutralization in the human population, while the AAV2 capsid peptide or viral particles exhibit a perceived higher seropositivity and resulting neutralization in the human population. Obviously, the specific seropositivity will depend on the population examined and the immunological methods used, but it has been reported that AAV8 exhibits a seropositivity of approximately 22% to approximately 38%, while AAV2 exhibits a seropositivity of approximately 43.5% to approximately 72%. See, e.g., Boutin et al., 2010, “Prevalence of serum IgG and neutralizing factors against AAV types 1, 2, 5, 6, 8 and 9 in the healthy population: implications for gene therapy using AAV vectors,” Hum. Gene Ther., 21:704-12. See also, Calcedo et al., 2009, J. Infect. Dis., 199:381-90.

[0117] Predicted adeno-associated virus (AAV) ancestral nucleic acid and polypeptide sequences

[0118] Multiple clones from libraries encoding ancestral shell polypeptides predicted from the Anc80 node were sequenced, and the amino acid sequences of representative AAV-predicted ancestral shell polypeptides are shown in SEQ ID NO: 19 (Anc80L27); SEQ ID NO: 20 (Anc80L59); SEQ ID NO: 21 (Anc80L60); SEQ ID NO: 22 (Anc80L62); SEQ ID NO: 23 (Anc80L65); SEQ ID NO: 24 (Anc80L33); SEQ ID NO: 25 (Anc80L36); and SEQ ID NO: 26 (Anc80L44). Those skilled in the art will understand that the nucleic acid sequence encoding each amino acid sequence can be readily determined.

[0119] In addition to the predicted ancestral shell polypeptide having the sequence shown in SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, or 26, a polypeptide is provided that has at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with the sequence shown in SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, or 26. Similarly, a nucleic acid is provided that has at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with the nucleic acid molecule encoding the ancestral shell polypeptide (i.e., having at least 95% sequence identity).

[0120] When calculating percentage sequence identity, two sequences are aligned and the number of identical matches of nucleotide or amino acid residues between them is determined. This number of identical matches is divided by the length of the aligned region (i.e., the number of aligned nucleotide or amino acid residues) and multiplied by 100 to obtain the percentage sequence identity value. It should be understood that the length of the aligned region can be a portion of one or two sequences up to the full length of the shortest sequence. It should also be understood that a single sequence can be aligned with more than one other sequence; therefore, different percentage sequence identity values ​​can be obtained relative to each aligned region.

[0121] The algorithm described by Altschul et al. (1997, Nucleic Acids Res., 25:3389 3402) can be used to align two or more sequences to determine the percentage sequence identity. This algorithm is incorporated into the BLAST (Basic Local Alignment Search Tool) program, which is available on the World Wide Web at ncbi.nlm.nih.gov. A BLAST search can be performed to determine the percentage sequence identity between a sequence (nucleic acid or amino acid) aligned using the algorithm of Altschul et al. and any other sequence or part thereof. BLASTN is a program for aligning and comparing the identity between nucleic acid sequences, while BLASTP is a program for aligning and comparing the identity between amino acid sequences. When using BLAST programs to calculate the percentage identity between one sequence and another, the default parameters of each program are typically used.

[0122] Representative comparison shown in Figure 4 and Figure 5A and 5B middle. Figure 4The comparison of ancestral AAV VP1 shell peptides named Anc80L65 (SEQ ID NO: 23), Anc80L27 (SEQ ID NO: 19), Anc80L33 (SEQ ID NO: 24), Anc80L36 (SEQ ID NO: 25), Anc80L44 (SEQ ID NO: 26), Anc80L59 (SEQ ID NO: 20), Anc80L60 (SEQ ID NO: 21), and Anc80L62 (SEQ ID NO: 22) is shown. Figure 4 The comparison shown in the figure confirms the predicted variants at each of the 11 sites, as well as a single nonsynonymous mutation at position 609E in Anc80L60 (SEQ ID NO: 21), which could be a clonal artifact. Figure 5A and 5B The images show ancestral AAV VP1 shell peptides (Anc80L65 (SEQ ID NO: 23), Anc80L27 (SEQ ID NO: 19), Anc80L33 (SEQ ID NO: 24), Anc80L36 (SEQ ID NO: 25), Anc80L60 (SEQ ID NO: 21), Anc80L62 (SEQ ID NO: 22), Anc80L44 (SEQ ID NO: 26) and Anc80L59 (SEQ ID NO: 20)) and contemporary AAV VP1 shell peptides (AAV8 (SEQ ID NO: 27), AAV9 (SEQ ID NO: 28), AAV6 (SEQ ID NO: 29), AAV1 (SEQ ID NO: 30), AAV2 (SEQ ID NO: 31), AAV3 (SEQ ID NO: 32), AAV3B (SEQ ID NO: 33), and AAV7 (SEQ ID NO: 20)). Comparison between 34). Figure 5A and 5B The comparison showed that the ancestral AAV sequence had approximately 85% to 91% sequence identity with contemporary AAV sequences.

[0123] Vectors containing nucleic acid molecules encoding polypeptides are also provided. Vectors (including expression vectors) are commercially available or can be produced using recombinant technologies. Vectors containing nucleic acid molecules may have one or more expression elements operatively linked to such nucleic acid molecules, and may also include sequences such as those encoding selectable markers (e.g., antibiotic resistance genes), and / or sequences that can be used in polypeptide purification (e.g., 6xHis tags). Expression elements include nucleic acid sequences that direct and regulate the expression of nucleic acid coding sequences. An example of an expression element is a promoter sequence. Expression elements may also include one or more of the following: introns, enhancer sequences, reaction elements, or inducible elements that regulate the expression of nucleic acid molecules. Expression elements may be of bacterial, yeast, insect, mammalian, or viral origin, and vectors may contain combinations of expression elements from different sources. As used herein, operative linking means that the expression element is positioned in the vector relative to the coding sequence in a manner that directs or regulates the expression of the coding sequence.

[0124] Nucleic acid molecules, such as those in vectors (e.g., expression vectors, viral vectors), can be introduced into host cells. The term "host cell" refers not only to the specific cell that has received the nucleic acid molecule but also to the progeny or potential progeny of such cells. Many suitable host cells are known to those skilled in the art; host cells can be prokaryotic cells (e.g., Escherichia coli) or eukaryotic cells (e.g., yeast cells, insect cells, plant cells, mammalian cells). Representative host cells may include, but are not limited to, A549, WEHI, 3T3, 10T1 / 2, 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 fibroblasts, hepatocytes, and myoblasts derived from mammals (including humans, monkeys, mice, rats, rabbits, and hamsters). Methods for introducing nucleic acid molecules into host cells are well known in the art and include, but are not limited to, calcium phosphate precipitation, electroporation, heat shock, lipid transfection, microinjection, and virus-mediated nucleic acid transfer (e.g., transduction).

[0125] Regarding polypeptides, "purified" refers to a polypeptide (i.e., a peptide or polypeptide) that has been isolated or purified from the cellular components that naturally accompany it. Typically, a polypeptide is considered "purified" when it contains at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or 99%) by weight of the polypeptide and naturally occurring molecules associated with it. Because chemically synthesized polypeptides are inherently isolated from their naturally accompanying components, synthetic polypeptides are considered "purified," but they can also be removed from the components used to synthesize the polypeptide (e.g., amino acid residues). Regarding nucleic acid molecules, "isolated" refers to a nucleic acid molecule isolated from other nucleic acid molecules that are normally associated with it in the genome. Furthermore, isolated nucleic acid molecules can include engineered nucleic acid molecules, such as recombinant or synthetic nucleic acid molecules.

[0126] Peptides can be obtained (e.g., purified) from natural sources (e.g., biological samples) using known methods such as DEAE ion exchange, gel filtration, and / or hydroxyapatite chromatography. Purified peptides can also be obtained, for example, by expressing nucleic acid molecules in an expression vector or by chemical synthesis. The purity of peptides can be measured using any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC. Similarly, nucleic acid molecules can be obtained (e.g., isolated) using conventional methods, such as, but not limited to, recombinant nucleic acid techniques (e.g., restriction enzyme digestion and ligation) or polymerase chain reaction (PCR; see, for example, PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995). Furthermore, isolated nucleic acid molecules can be chemically synthesized.

[0127] Using ancestral viruses or parts thereof

[0128] Ancestor viruses or portions thereof, as described herein, particularly those exhibiting a reduced seropositivity rate relative to contemporary viruses or portions thereof, can be used in a wide range of research and / or therapeutic applications. For example, ancestral viruses or portions thereof, as described herein, can be used in human or animal medicine for gene therapy (e.g., for vectors or vector systems for gene transfer) or for vaccination (e.g., for antigen presentation). More specifically, ancestral viruses or portions thereof, as described herein, can be used for gene addition, gene augmentation, genetic delivery of peptide therapeutics, 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.

[0129] Host cells can be transduced or infected in vitro (e.g., grown in culture) or in vivo (e.g., in a subject) using ancestral viruses or portions thereof. This document describes host cells that can be transduced or infected in vitro using ancestral viruses or portions thereof; and host cell packages that can be transduced or infected in vivo using ancestral viruses or portions thereof, including, but not limited to, 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 gland).

[0130] The ancestral virus, as described herein, or a portion thereof, may be modified to include a transgene (in cis or trans form with other viral sequences). The transgene may be, for example, a reporter gene (e.g., β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymkinase, green fluorescent peptide (GFP), chloramphenicol acetyltransferase (CAT), or luciferase, or a fusion peptide including an antigen-tagged domain such as hemagglutinin or Myc) or a therapeutic gene (e.g., a gene encoding a hormone or its receptor, a growth factor or its receptor, a differentiation factor or its receptor, an immune system regulator (e.g., cytokines and interleukins) or its receptor, an enzyme, RNA (e.g., repressive RNA or catalytic RNA), or a target antigen (e.g., a carcinogenic antigen, an autoimmune antigen)).

[0131] Specific gene transfers will depend at least in part on the specific disease or defect being treated. For example, gene transfer or gene therapy may be applied to treat the following diseases: hemophilia, retinitis pigmentosa, cystic fibrosis, leber congenital amaurosis, lysosomal storage diseases, and 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 degeneration (e.g., age-related macular degeneration), lipoprotein 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 syndrome, fatal familial insomnia, Creutzfeldt-Jakob disease, sickle cell disease. Diseases including Huntington's disease, fronto-temporal lobar degeneration, Usher syndrome, lactose intolerance, lipid storage disorder (such as Niemann-Pick type C), Batten's disease, choroideremia, glycogen storage disease type II (Pompedis disease), ataxia telangiectasia (Louis-Bar syndrome), congenital hypothyroidism, severe combined immunodeficiency (SCID), and / or amyotrophic lateral sclerosis (ALS).

[0132] Transgenic organisms can also be, for example, immunogens that can be used to immunize subjects (e.g., humans, animals (e.g., companion animals, farm animals, endangered animals)). For example, immunogens can be derived from organisms (e.g., pathogenic organisms) or their immunogenic parts or components (e.g., toxic peptides or their byproducts). Pathogenic organisms from which immunogenic peptides can be obtained, for example, include viruses (e.g., parvoviruses, enteroviruses, orthomyxoviruses, reoviruses, retroviruses), prokaryotes (e.g., pneumococci, staphylococci, Listeria, Pseudomonas) and eukaryotes (e.g., amebiasis, malaria, leishmaniasis, nematodes). It will be understood that the methods described herein and the compositions produced by such methods are not limited to any specific transgenic organism.

[0133] An ancestral virus or a portion thereof, typically suspended in a physiologically compatible vector, can be administered to a subject (e.g., a human or non-human mammal). Suitable vectors include saline (which can be prepared using various buffer solutions, such as phosphate-buffered saline), lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, and water. The ancestral virus or a portion thereof is administered in sufficient quantities to transduce or infect cells and to provide adequate levels of gene transfer and expression to deliver therapeutic benefit without inappropriate side effects. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to organs such as, for example, the liver or lungs, orally, intranasally, intratracheally, by inhalation, intravenously, intramuscularly, intraocularly, subcutaneously, intradermally, across mucous membranes, or via other routes of administration. Routes of administration may be combined as needed.

[0134] The dosage of the ancestral virus or a portion thereof administered to the subject will depend primarily on factors such as the condition being treated, as well as the subject's age, weight, and health. For example, the therapeutically effective dose of the ancestral virus or a portion thereof to be administered to human subjects is typically in the range of approximately 0.1 ml to approximately 10 ml of solution containing approximately 1 × 10⁻⁶ ppm. 1 Up to 1×10 12 The concentration of ancestral virus genome copies (GCS) (e.g., approximately 1 × 10⁻⁶) 3 Up to 1×10 9 Transduction and / or expression of the transgene can be monitored at multiple time points post-administration using DNA, RNA, or protein assays. In some cases, transgene expression levels can be monitored to determine the frequency and / or amount of the dose. Dosing regimens similar to those described for therapeutic purposes can also be used for immunization.

[0135] The method described in this paper can also be used to model forward evolution in order to modify or remove one or more immunogenic domains of a virus or a portion thereof.

[0136] According to the present invention, conventional molecular biology, microbiology, biochemistry, and recombinant DNA techniques within the scope of the art can be employed. These techniques are fully explained in the literature. The invention will be further described in the following examples, which do not limit the scope of the methods and compositions described in the claims.

[0137] Example

[0138] Example 1: Computer Prediction of Ancestor Sequences

[0139] A set of 75 different amino acid sequences of the AAV shell were obtained from many public databases (including GenBank) and the sequences were aligned using the PRANK-MSA algorithm, version 121002, with the option "-F".

[0140] ProtTest3 (see, for example, Darriba et al., 2011, Bioinformatics, 27(8):1164-5; available on the World Wide Web at darwin.uvigo.es / software / prottest3) was used to evaluate different models of peptide evolution under different conditions (e.g., those included in ProTest3, i.e., “+I”, “+F”, “+G” and combinations thereof) (e.g., those models included in ProTest3, i.e., JTT, LG, WAG, VT, CpRev, RtRev, Dayhoff, DCMut, FLU, Blosum62, VT, HIVb, MtArt, MtMam). The JTT model was selected based on the scores of the Aikka Information Criterion (AIC; Hirotugu, 1974, IEEE Transactions on Automatic Control, 19:716-23) implemented in ProTest3 (Jones et al., 1992, Comp. Appl. Biosci., 8:275-82) with +G and +F (Yang, 1993, Mol. Biol. Evol., 10:1396-1401; and Cao et al., 1994, J. Mol. Evol., 39:519-27).

[0141] Phylogenetic analysis of AAV evolution was constructed using PhyML (Guindon and Gascuel, 2003, Systematic Biology, 52:696-704). See also Figure 3 Trees were generated using a JTT+ F substitution model with four discrete substitution categories and estimated Gamma-shaped parameters. The resulting trees were improved by Nearest Neighbor Interchange (NNI), Subtree Pruning, and SPR, and significance was assessed using the “SH-Like” variant using bootstrap and approximate likelihood ratio test (aLRT; Anisimova and Gascuel, 2006, Systematic Biology, 55:539-52).

[0142] Then, the constructed phylogenetic tree was used to estimate the ancestral state of the AAV shell at each node within the phylogenetic tree. The ancestral shell sequence was reconstructed using the maximum likelihood principle via the Maximum Likelihood Phylogenetic Analysis by Maximum Likelihood (PAML) software included in Lazarus (Sourceforge at sf.net) (Yang, 1997, Comp. Applic. BioSci., 13:555-6; available at abacus.gene.ucl.ac.uk / software / paml.html). More specifically, Lazarus / PAML reconstruction was set up, using a JTT+F substitution model with a 4-gamma distribution class to produce the amino acid reconstruction. AAV5 was used as the outgroup. Finally, an "I" option was added to place insertions and deletions (i.e., binary encodings placed using the Fitch algorithm with maximum parsimony) after the PAML reconstruction was completed.

[0143] Because reconstruction is performed in a maximum likelihood manner, the probability of any residue at a given position at a given node can be calculated. To do this, additional scripts were written to identify all positions along the sequence with a calculated posterior probability below a certain threshold. A threshold of 0.3 was chosen, meaning that any amino acid with a calculated posterior probability greater than 0.3 would be included in the synthesis of the library. These residues were then selected as variants of interest in the library.

[0144] To finalize the sequence, an additional utility tool for codon selection must be encoded. A script was written to derive codons similar to those of another AAV sequence (AVVRh10, which has approximately 92% sequence identity with the Anc80 scaffold sequence) and apply a new algorithm to replace the codons where sequence mismatches exist based on the codon replacement matrix. The new algorithm is shown below:

[0145] Given: an amino acid sequence, Pt, with a corresponding nucleotide sequence, NT, where Nt encodes Pt; and a protein sequence, Pi, where Pi exhibits strong homology with Pt.

[0146] Pi and Pt were aligned using the Needleman-Wunsch Blosum62 table for scoring. New nucleotide sequences, Ni, were generated by stepping the protein alignment using the corresponding codons from Nt.

[0147] The amino acids in Pt are a perfect match for those in Pi.

[0148] The "best score" codon from the codon-PAM matrix (Schneider et al., 2005, BMCBioinform., 6:134) has substitutions.

[0149] A gap exists in Pi, where the amino acids in Pt are compared, and

[0150] The most frequently occurring nucleotide in Nt (encoding a given amino acid) is the amino acid present in Pi, which corresponds to the gap in Pt.

[0151] In addition, two single nucleotide changes were generated to eliminate transcription of the assembly-activating protein (AAP), which is encoded out of frame within the AAV capsid gene in wild-type AAV. Since the encoding of AAP (current or ancestral) is not part of this reconstruction, AAP expression was eliminated by generating a synonymous mutation in the cap sequence, and the AAP sequence was provided in trans form during viral production.

[0152] Example 2: Expression of ancestral AAV VP1 sequence

[0153] Experiments were conducted to determine whether the predicted ancestral AAV capsid sequence could be used to prepare viral vectors.

[0154] Numerous predicted ancestral AAV capsid sequences were cloned. The ancestral capsid library was transferred to the rep-cap expression plasmid, enabling viral particle formation during transient transfection. To maintain appropriate expression levels and splicing of VP1, VP2, and VP3, the library cap gene was cloned by cleaving the 5' HindIII at the cap coding sequence in the rep and a SpeI engineered between the cap stop codon and the polyadenylation signal. Therefore, to clone the ancestral capsid into a more conventional “REP / CAP” construct, the passaged plasmid was digested with HindIII and SpeI, purified by gel electrophoresis, and ligated into a similarly digested rep / cap plasmid.

[0155] The expressed peptides were resolved on 10% SDS gels. For example... Figure 6 As shown, the shell polypeptide is appropriately expressed and spliced ​​from multiple ancestral AAV sequences (Anc80L44, Anc80L27 and Anc80L65) as well as from contemporary AAV sequences, AAV2 / 8, into VP1, VP2 and VP3.

[0156] Example 3: Virus Titration

[0157] AAVs were generated in HEK293 cells by transient co-transfection with plasmids encoding all the elements required for viral particle assembly. In short, HEK293 cells were cultured to 90% confluence and transfected with the following: (a) a viral genome plasmid encoding a luciferase transgene flanking AAV2 ITR (expressed via a CMV promoter), (b) an AAV packaging plasmid encoding the AAV2 rep and synthetic capsid protein disclosed herein, (c) an AAV2-AAP capsid expression, and (d) an adenovirus helper gene required for AAV packaging and assembly. Cells were cultured at 37°C for 2 days, and cells and culture medium were harvested and collected.

[0158] The cell culture medium suspension was lysed by three consecutive freeze-thaw cycles. The lysate was then clarified by centrifugation and subjected to complete DNA digestion using an enzyme (Benzonase was used in this study). TM The AAV preparation is processed to digest any DNA present outside the viral particles. The AAV preparation is diluted to fall within the linear measurement range of the control DNA template, in this case, a linearized plasmid having the same TaqMan™ primer and probe binding sequences as the vector genome. TaqMan™ PCR is performed using primers and probes annealed to the selected viral vector genome. Titers are calculated based on TaqMan™ measurements in genomic copies per milliliter (ml), as shown in Table 2 below.

[0159] Table 2

[0160]

[0161] Small-scale carrier production results on ancestor-reconstructed AAV shell particles showed similarities to AAV2, but with reduced yields compared to AAV8 (both of which are carrier preparations based on contemporary AAV).

[0162] Example 4: In vitro viral transduction

[0163] In vitro viral transduction was performed to evaluate the ability of viruses containing predicted ancestral AAV sequences to infect cells.

[0164] Following high-throughput vector production using the Anc80 sequence library, HEK293 cell lines were transduced with each viral vector. Each viral vector contained a luciferase transgene in addition to the Anc80 sequence. Luciferase was quantitatively measured using bioluminescence in a 96-well plate reader after the luciferin substrate was added to the transduced cells or cell lysates. Following quantification, heatmaps of luciferase expression were generated in four cascaded 96-well plates (excluding the control column in each plate). Due to the numerous insertions, deletions, and conversions associated with the high-throughput vector production process, many vectors were non-functional. For the purposes of this study, only the functional viruses that were capable of transducing HEK293 cells and expressing the transgene were further evaluated in this assay.

[0165] Two contemporary AAV vectors (AAV2 / 2 and AAV2 / 8) and three predicted ancestral AAV vectors (Anc80L27, Anc80L44, and Anc80L65) were used at a rate of 1 × 10⁻⁶ cells per cell. 4 HEK293 cells were transduced with an equal number of genome copies (GCs) of multiple infection (MOI). Each vector contained either a luciferase-encoded transgene or an eGFP-encoded transgene. Cells were imaged using the GFP channel of an AMG EvosFl optical microscope after 60 hours. Figure 7 The expression of luciferase is shown after in vitro transduction. Each ancestral AAV virus indicates efficient transduction of HEK293 cells.

[0166] Example 5 - In vivo retinal transduction

[0167] Retinal transduction was performed to determine whether the ancestral AAV vector could target mouse retinal cells in vivo.

[0168] Use 2×10 8Mouse eyes were transduced with three distinct ancestral AAVs (Anc80L27, Anc80L44, and Anc80L65) and contemporary AAVs (AAV2 / 8), all of which included eGFP-encoded transgenes. For transduction, each AAV vector was surgically delivered subretinal by vector bolus delivery via an injection device, creating a space between the photoreceptor and the retinal pigment epithelium. The vector bolus was left in the subretinal space, and retinal detachment was resolved over time. GFP expression was non-invasively monitored by bottom photography of the animal retina after pupil dilation using Tropicamide™. All presented retinas indicated varying degrees of successful targeting of the ancestral AAVs to the retina.

[0169] Retinal histology was also performed and visualized under fluorescence microscopy to identify the transduced cell types. Histology was performed on mouse retinas transduced with the Anc80L65 ancestral AAV vector as described above. Anc80L65-mediated eGFP expression was evident in the outer nuclear layer (ONL), inner segments (IS), and retinal pigment epithelium (RPE), indicating that the ancestral Anc80L65 vector targets mouse photoreceptors and retinal pigment epithelial cells.

[0170] Example 6: Neutralizing Antibody Assay

[0171] Perform a neutralizing antibody assay to assess whether ancestral AAV viruses are more resistant to antibody neutralization than contemporary AAV viruses. The neutralizing antibody assay measures the concentration of antibodies (or the titer of antibody concentration contained in the experimental sample) that neutralizes infection by 50% or more compared to a control without antibodies.

[0172] Serum samples or IVIG stock solutions were serially diluted 2-fold, and undiluted and diluted samples were mixed with 10... 4The ancestral AAV virus of MOI, Anc80L65, and the contemporary AAV virus, AAV2 / 8, were co-incubated at 37°C for approximately 30 minutes. Each virus contains a luciferase gene. The mixed vector and antibody sample were then transduced into HEK293 cells. For these experiments, the antibody sample used was intravenous immunoglobulin (IVIG), a pooled IgG extracted from the plasma of over 1000 blood donors (commercially available, e.g., Gammagard™ (Baxter Healthcare; Deerfield, IL) or Gamunex™ (Grifols; LosAngeles, CA)). Luciferase was detected in the cells by bioluminescence assay 48 hours after transduction initiation. Neutralizing antibody titers were determined by identifying the dilution of the sample that achieved 50% or greater neutralization (sample transduction / control virus transduction in the absence of the sample).

[0173] like Figure 8 As shown, to reduce the transduction efficiency of the ancestral AAV virus Anc80L65 to below 50% in the IVIG-free control (dashed line), a significantly higher concentration of IVIG is required compared to the contemporary AAV virus AAV2 / 8. These results demonstrate the higher resistance of the ancestral AAV virus to IVIG neutralization compared to contemporary AAV viruses.

[0174] Example 7: Characterization of Anc80

[0175] Following the methods described herein, the most probable Anc80 sequence (as determined by posterior probability) was obtained and designated Anc80L1 (SEQ ID NO: 35 shows the nucleic acid sequence of the Anc80L1 shell and SEQ ID NO: 36 shows the amino acid sequence of the Anc80L1 VP1 polypeptide). An Anc80 probabilistic libraries were also synthesized using the sequences described herein and subcloned into expression vectors by a commercial company.

[0176] In a combined assay, the vector yield and infectivity of the Anc80 library were evaluated by cloning. Anc80L65 (SEQ ID NO: 23) and several other variants were further characterized during screening.

[0177] The Anc80 library and Anc80L65 were compared in terms of sequence differences. Figure 9 ; from diagonally upwards, % for amino acid differences below (#). Using NCBI-BLAST, the most closely related publicly available sequence to Anc80L65 is rh10 (GenBank accession number AAO88201.1).

[0178] Figure 10The results show that Anc80L65 produced a vector yield comparable to AAV2 (Fig. A), generated viral particles under transmission electron microscopy (TEM) (Fig. B), and biochemically produced AAV cap and VP1, 2, and 3 proteins based on SDSPAGE under denaturing conditions (Fig. C) and Western blotting using AAV capsid antibody B1 (Fig. D). These experiments are described in more detail in the following paragraphs.

[0179] Briefly, AAV2 / 8, AAV2 / 2, AAV2 / Anc80L27, AAV2 / Anc80L44, and AAV2 / Anc80L65 vectors containing reporter constructs consisting of eGFP under CMV promoter control and firefly luciferase were produced on a small scale. The titers of these small-scale viral preparations were then obtained by qPCR. Based on these experiments, the Anc80L27, Anc80L44, and Anc80L65 vectors were found to produce viral levels comparable to those of AAV2. Figure 10 (A).

[0180] To confirm the assembly of the Anc80L65 capsid protein into complete virus-like particles of appropriate size and conformation, transmission electron microscopy (TEM) was used to obtain micrographs. Large-scale purified Anc80-L065 preparations were loaded onto polyvinyl formal (Formvar®)-coated copper grids and stained with uranylacetate. The micrographs revealed complete, hexagonal particles with diameters between 20 and 25 nm. Figure 10 (B).

[0181] To determine whether the synthesized ancestral capsid genes had been properly processed (i.e., spliced ​​and expressed), large-scale purified preparations of AAV2 / 8, AAV2 / 2, and AAV2 / Anc80L65 vectors were loaded onto SDS-PAGE gels (1E10 GC / well) under denaturing conditions. For each vector preparation, bands representing the viral capsid proteins VP1, VP2, and VP3 were clearly present. Figure 10 The C). Western blotting using AAV capsid antibody B1 further confirmed that these bands represented the predicted protein (C). Figure 10 (D).

[0182] also, Figure 11The results show that, relative to AAV2 and / or AAV8 controls, using GFP as the readout (Fig. A) or luciferase (Fig. B), Anc80L65 infected mammalian tissues and cells in vitro on HEK293 cells at an MOI of 10E4GC / cell. Following IV injection of the designated AAV encoding the nuclear LacZ transgene (top row, Fig. C), direct intramuscular (IM) injection of the designated AAV encoding GFP (middle row, Fig. C), and subretinal injection of the designated AAV encoding GFP (bottom row, Fig. C), Anc80L65 also effectively targeted the liver. These experiments are described in more detail in the following paragraphs.

[0183] To obtain a relative measurement of the infectivity of the ancestral virion, crude preparations containing bicistronic reporter constructs AAV2 / 2, AAV2 / 8, AAV2 / Anc80L65, AAV2 / Anc80L44, AAV2 / Anc80L27, AAV2 / Anc80L121, AAV2 / Anc80L122, AAV2 / Anc80L123, AAV2 / Anc80L124, and AAV2 / Anc80L125 were produced, wherein the bicistronic reporter constructs comprise eGFP and firefly luciferase under the control of the CMV promoter. Each vector was then transduced into 96-well plates confluent with HEK293 cells at an MOI of 1E4 GC / cell (titer obtained by qPCR as described above). After 48 hours, fluorescence microscopy confirmed the presence of GFP in the transduced cells. Figure 11 A). Then the presence of luciferase in the cells was measured ( Figure 11 (B) This determined that the expression of luciferase in cells transduced with Anc80-derived vectors was between that in cells transduced with AAV8 (lower levels of transduction) and in cells transduced with AAV2 (higher levels of transduction).

[0184] To assess the relative efficiency of gene transfer in vivo, high-titer preparations of purified AAV2 / 2, AAV2 / 8, and AAV2 / Anc80L65 were obtained. Under general anesthesia, each vector (with capsidation of a transgene encoding the nuclear LacZ under TBG promoter control) of 3.9E10 GC was injected via intraperitoneal injection into C57BL / 6 mice (3 mice per condition). Twenty-eight days post-injection, mice were sacrificed and tissues were collected. Liver sections were prepared using standard histological techniques and stained against β-galactosidase. The sections were then imaged under a microscope, and representative images are shown below. Figure 11 C, top row.

[0185] Vectors of the same serotype were then obtained, containing bicistronic transgenes encoding eGFP and hA1AT under the control of the pCASI promoter. To assess the ability of Anc80L65 to transduce mouse skeletal muscle, each vector of 1E10 GC was injected into the skeletal muscle of C57BL / 6 mice (5 mice per condition) under general anesthesia. Mice were sacrificed 28 days post-injection, tissue sections were frozen, and the presence of eGFP was assessed using fluorescence confocal microscopy (blue for DAPI, green for eGFP). Representative images are shown in [image description missing]. Figure 11 The C in the middle row. These experiments show that the Anc80L65 vector can transduce mouse skeletal muscle via intramuscular injection.

[0186] Vectors of the same serotype were obtained, and this time, a construct encoding only the eGFP transgene was created under CMV promoter control. The 2E9 particles were subretinally injected into C57BL / 6 mice under general anesthesia. Mice were sacrificed 28 days post-injection, and eyes were collected, frozen sections were prepared, and the presence of eGFP was assessed using fluorescence confocal microscopy (blue for DAPI, green for eGFP). Representative images are shown in [image description missing]. Figure 11 The C-line of the bottom row. These experiments show that the Anc80L65 vector can transduce the mouse retina at a level comparable to that of the AAV8 vector.

[0187] Figure 12 The experimental results are shown, in which the seropositivity rate of the ancestral viral vector was assessed relative to existing AAV viral vectors. Using an in vitro neutralizing antibody assay, Anc80L65 exhibited increased resistance to neutralization using IVIG (pharmaceutical confluence serum from approximately 10,000 individuals). Figure 12 A). Additionally, in the whisker box diagram, serum from Belgian individuals (n=100; Figure 1 2C) or serum from Boston individuals (n=102); Figure 12 B) showed reduced sensitivity (or increased resistance) to neutralizing Anc80L65 relative to AAV2,8, and similar seropositivity relative to rh32.33 (a different AVV vector with a known minimum seropositivity rate but limited use as a gene therapy vector). Serum obtained from cynomolgus monkeys showed similar increased resistance compared to AAV2,8 and rh32.33. Figure 12 (D). These experiments are described in more detail in the following paragraphs.

[0188] In summary, purified high-titer preparations of the AAV2 / 8, AAV2 / 2, AAV2 / rh32.33, and AAV2 / Anc80L65 viral vectors were obtained, with capsidation comprising bicistron transgenesis of eGFP and firefly luciferase under CMV promoter control. These vectors were then incubated with or without sequentially diluted IVIG (1E9 GC for each condition). After incubation, the vectors were transduced into HEK293 cells at 1E4 MOI per well (one dilution per well). The relative amount of luciferase was determined by luminescence assay after 48 hours. Transduction relative to a serum-free control is shown in [data missing]. Figure 12 In A. Serum from individuals in Belgium (n=100; Figure 12 C) or serum from Boston individuals (n = 102; Figure 12 A similar experiment was performed in B). For each serum sample, the neutralization titer was reported as a 50% reduction in dilution relative to the serum-free control, given the carrier. Figure 12 Neutralizing titers were reported in boxes and whiskers plots in B and C, which determined that the Anc80L65 vector had lower prevalence in both populations (Belgian or Boston populations) compared to AAV2 and AAV8, approaching the level of Rh32.33 (different AAV vectors with the lowest known seropositivity). Serum obtained from cynomolgus monkeys was evaluated in the same manner. Figure 12 The study found that Anc80L65 had a significantly lower seropositivity rate compared to AAV2, AAV8, and Rh.32.33 vectors.

[0189] Example 8: Generation of other ancestral AAV shells

[0190] The most probable ancestral AAV shell sequences were then synthesized in a commercial laboratory (Gen9) (as determined by posterior probability) and provided as linear dsDNA. These amino acid sequences were then compared with those of extant AAVs to determine the extent of their differences (Figure 13). Each ancestral VP1 protein differed from the VP1 protein of a selected representative extant AAV by 3.6%–9.3% (…). Figure 13A The difference between the ancestral VP3 protein and the VP3 protein is 4.2%-9.4%. Figure 13B These shells were individually subcloned into AAV production plasmids (pAAVector2 / empty) by restriction enzyme digestion (HindIII & SpeI) and T4 ligation. These clones were confirmed by restriction digestion and Sanger sequencing, resulting in a medium-sized preparation of plasmid DNA.

[0191] Each of these plasmids was then used to generate an AAV vector containing a reporter gene encoding both eGFP and firefly luciferase. These vectors were generated in triplicate on a small scale, as previously described. The crude viral preparation was then titrated by qPCR and found to produce 2.71% to 183.1% of viral particles relative to AAV8. Figure 14 and 15 These titers were then used to establish titer control experiments to assess relative infectivity. Anc126 had no titer control due to its significantly reduced production; therefore, data on the infectivity of Anc126 cannot be accurately compared to the infectivity of other viruses used in the experiments. Other vectors were used to transduce HEK293 cells at a multiple infection (MOI) rate of 1.9E3 GC / cell.

[0192] Sixty hours after transduction, GFP expression in cells was assessed by fluorescence microscopy. Except for the negative control, eGFP-positive cells were detected under each condition. Figure 16 This indicates that each synthesized and cloned ancestral sequence was predicted to produce viable, infectious viral particles. To obtain an idea of ​​the relative levels of infectivity, a luciferase assay was also performed on the same cells. The results indicated that each ancestral vector was able to transduce 28.3% to 850.8% of HEK293 cells relative to AAV8. Figure 17 and 18 It should be noted that Anc126 was excluded from the analysis of relative transduction because it had no titer control.

[0193] In summary, eight novel ancestral AAV capsid genes were synthesized and used in conjunction with AAV8, AAV2, and the previously described Anc80L65 vector to generate functional viral vectors. Production and infection were evaluated in vitro, and the findings are summarized in [the table / document / etc.]. Figure 19 .

[0194] Example 9: Vector-based Immunoprophylaxis

[0195] In vectored immunoprophylaxis, gene therapy vectors (such as AAVs) are used to deliver transgenes encoding broad-spectrum neutralizing antibodies against infectious agents. See, for example, Balazs et al. (2013, Nat. Biotechnol.,31:647-52); Limberis et al. (2013, Sci. Transl. Med., 5:187ra72); Balazs et al. (2012, Nature, 481:81-4); and Deal et al. (2014, PNAS USA, 111:12528-32). One advantage of this treatment is that the host produces antibodies in its own cells, meaning that a single administration has the potential to confer lifelong protection against the pathogen (etiologic agent).

[0196] Example 10: Drug Delivery Carrier

[0197] LUCENTIS® (ranibizumab) and AVASTIN® (bevacizumab) are both anti-angiogenic agents based on the same humanized mouse monoclonal antibody against vascular endothelial growth factor A (VEGF-A). Although bevacizumab is a complete antibody and ranibizumab is a fragment (Fab), they work through the same mechanism—by antagonizing VEGF—to treat wet age-related macular degeneration. See, for example, Mao et al. (2011, Hum. Gene Ther., 22:1525-35); Xie et al. (2014, Gynecol. Oncol., doi: 10.1016 / j.ygyno.2014.07.105); and Watanabe et al. (2010, Gene Ther., 17:1042-51). Since both molecules are proteins, they can be encoded by DNA and produced in cells transduced with a transgenic vector, and are small enough to be packaged into an AAV vector.

[0198] Other implementation plans

[0199] It should be understood that although the methods and material compositions have been described herein in conjunction with several different aspects, the foregoing description is intended to illustrate, not limit, the scope of the methods and material compositions. Other aspects, advantages, and modifications fall within the scope of the appended claims.

[0200] This document discloses methods and compositions that can be used in conjunction with the products of the disclosed methods and compositions, and can be used to prepare the products of the disclosed methods and compositions or the products of the disclosed methods and compositions. These and other substances are disclosed herein, and it should be understood that combinations, subgroups, interactions, groups, etc., of these methods and compositions are disclosed. That is, while every different individual and collective combination and substitution of these compositions and methods may not be explicitly disclosed, each is specifically considered and described herein. For example, if a particular composition of substances or a particular method is disclosed and discussed, and multiple compositions and methods are discussed, then every combination and substitution of compositions and methods is explicitly covered unless explicitly indicated to the contrary. Similarly, any subgroups or combinations thereof are also explicitly covered and disclosed.

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Claims

1. An adeno-associated virus (AAV) capsid polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 and 17.

2. The AAV capsid polypeptide of claim 1, wherein the AAV capsid polypeptide or viral particles containing the AAV capsid polypeptide exhibit a lower seroprevalence than the AAV2 capsid polypeptide or viral particles containing the AAV2 capsid polypeptide, and wherein the AAV capsid polypeptide or viral particles containing the AAV capsid polypeptide exhibits substantially the same or lower seroprevalence than the AAV8 capsid polypeptide or viral particles containing the AAV8 capsid polypeptide.

3. The AAV capsid polypeptide of claim 1, wherein the AAV capsid polypeptide or viral particles containing the AAV capsid polypeptide are neutralized by human serum to a lesser extent than the AAV2 capsid polypeptide or viral particles containing the AAV2 capsid polypeptide, and wherein the AAV capsid polypeptide or viral particles containing the AAV capsid polypeptide are neutralized by human serum to a similar or lesser extent than the AAV8 capsid polypeptide or viral particles containing the AAV8 capsid polypeptide.

4. The AAV shell polypeptide of any one of claims 1-3, wherein the AAV shell polypeptide is purified.

5. The AAV shell polypeptide of claim 1, encoded by a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 and 18.

6. A nucleic acid molecule encoding an adeno-associated virus (AAV) capsid polypeptide having a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 and 18.

7. A vector comprising the nucleic acid molecule of claim 6.

8. A host cell comprising the vector of claim 7.

9. Purified viral particles comprising the AAV capsid polypeptide of any one of claims 1-5.

10. The purified virus particles of claim 9, further comprising genetic material.

11. An adeno-associated virus (AAV) capsid polypeptide having at least 95% sequence identity with an amino acid sequence selected from the group consisting of: SEQ ID NO: 19, 20, 21, 22, 23, 24, 25 and 26.

12. The AAV capsid polypeptide of claim 11, wherein the AAV capsid polypeptide or viral particles containing the AAV capsid polypeptide exhibit a lower seropositivity rate than the AAV2 capsid polypeptide or viral particles containing the AAV2 capsid polypeptide, and wherein the AAV capsid polypeptide or viral particles containing the AAV capsid polypeptide exhibits substantially the same or lower seropositivity rate than the AAV8 capsid polypeptide or viral particles containing the AAV8 capsid polypeptide.

13. The AAV capsid polypeptide of claim 11, wherein the AAV capsid polypeptide or viral particles containing the AAV capsid polypeptide are neutralized by human serum to a lesser extent than the AAV2 capsid polypeptide or viral particles containing the AAV2 capsid polypeptide, and wherein the AAV capsid polypeptide or viral particles containing the AAV capsid polypeptide are neutralized by human serum to a similar or lesser extent than the AAV8 capsid polypeptide or viral particles containing the AAV8 capsid polypeptide.

14. The AAV shell polypeptide of any one of claims 11-13, wherein the AAV shell polypeptide is purified.

15. The AAV shell polypeptide of any one of claims 11-14, wherein the polypeptide has at least 99% sequence identity with an amino acid sequence selected from the group consisting of: SEQ ID NO: 19, 20, 21, 22, 23, 24, 25 and 26.

16. The AAV shell polypeptide of any one of claims 11-14, wherein the polypeptide has 100% sequence identity with an amino acid sequence selected from the group consisting of: SEQ ID NO: 19, 20, 21, 22, 23, 24, 25 and 26.

17. A viral particle comprising at least one of the AAV capsid polypeptides of any one of claims 11-16.

18. The virus particle of claim 17, further comprising genetic material.

19. A method for gene transfer or vaccination using transgenic technology, said method comprising: The viral particles of claim 10 or claim 18 are administered to subjects requiring gene transfer or immunization, wherein the viral particles exhibit a lower seropositivity rate than AAV2 viral particles.

20. The method of claim 19, wherein the viral particles exhibit substantially the same or lower seropositivity rate as the AAV8 viral particles.

21. The method of claim 19, wherein the virus particles are neutralized by human serum to a lesser extent than AAV2 virus particles, and wherein the AAV virus particles are neutralized by human serum to a similar or lesser extent than AAV8 virus particles.

22. A method for administering a vaccine to a subject, the method comprising: The target antigen of the AAV shell polypeptide operably linked to claim 1 or claim 11 is administered to a subject in need of vaccination, wherein the AAV shell polypeptide exhibits a lower seropositivity rate than the AAV2 shell polypeptide.

23. The method of claim 22, wherein the AAV shell polypeptide exhibits substantially the same or lower seropositivity rate as the AAV8 shell polypeptide.

24. The method of claim 22, wherein the AAV shell polypeptide is neutralized by human serum to a lesser extent than the AAV2 shell polypeptide, and the AAV shell polypeptide is neutralized by human serum to a similar or lesser extent than the AAV8 shell polypeptide.

25. A computer method (in silico method) for predicting the sequence of an ancestral virus or a portion thereof, the method comprising: Provide nucleotide or amino acid sequences from multiple contemporary viruses or portions thereof; The sequences were compared using the Multiple Sequence Alignment (MSA) algorithm; Evolutionary modeling is used to obtain predicted ancestral phylogenetic relationships for the multiple contemporary viruses or parts thereof; At the phylogenetic node of the predicted ancestral phylogeny, the evolutionary probability of a specific nucleotide or amino acid residue at each position in the sequence is estimated, and Predict the sequence of the ancestral virus or a portion thereof based on the estimated probability at each position.

26. The method of claim 25, wherein all steps are performed using a computer processor.

27. The method of claim 25 or claim 26, wherein the MSA algorithm uses phylogenetic information to predict whether a gap in the alignment is the result of a missing or inserted object.

28. The method of claim 27, wherein the MSA algorithm is a Probabilistic Alignment Kit (PRANK).

29. The method of any one of claims 25 to 28, wherein the model used for evolutionary modeling is selected using the Aikake Information Criterion (AIC).

30. The method of any one of claims 25 to 28, wherein the predicted ancestral phylogeny is obtained using the JTT model and the gamma distribution model ("+ G") and the frequency calculation of πi ("+ F").

31. The method of claim 25, wherein the step of modeling evolution is performed using the JTT+G+F model.

32. The method of any one of claims 25 to 31, further comprising synthesizing the ancestral virus or a portion thereof based on the predicted sequence.

33. The method of claim 32, further comprising assembling the ancestral virus or a portion thereof into ancestral virus particles.

34. The method of claim 33, further comprising screening the ancestral virus particles for at least one of the following: (1) replication; (b) gene transfer properties; (c) receptor binding; or (d) seropositivity.

35. The method of claim 34, wherein the ancestral virus particles exhibit a lower seropositivity rate than virus particles assembled from at least one of the plurality of contemporary viruses or portions thereof.

36. The method of claim 34, wherein the ancestral virus particles are neutralized by human serum to a lesser extent than virus particles assembled from at least one of the plurality of contemporary viruses or portions thereof.

37. The method of claim 25, wherein the plurality of contemporary viruses or portions thereof belong to a family selected from the group consisting of: adenovirus (AV), human immunodeficiency virus (HIV), retrovirus, lentivirus, herpes simplex virus (HSV), vaccinia virus, pox virus, influenza virus, respiratory syncytial virus, parainfluenza virus, and foamy virus.

38. Use of the virus particles of claim 10 or claim 18 in the preparation of a composition for use in a method of gene transfer or vaccination with transgenic technology, the method comprising: The viral particles of claim 10 or claim 18 are administered to subjects who require gene transfer or immunization.

39. The use of claim 38, wherein the virus particles exhibit a lower seropositivity rate than AAV2 virus particles or the virus particles exhibit substantially the same or lower seropositivity rate as AAV8 virus particles.

40. The use of claim 38, wherein the virus particles are neutralized by human serum to a lesser extent than AAV2 virus particles, and wherein the AAV virus particles are neutralized by human serum to a similar or lesser extent than AAV8 virus particles.

41. Use of a target antigen operatively linked to the AAV shell polypeptide of claim 1 or claim 11 in the preparation of a composition for use in a method of vaccinating a subject, the method comprising: The target antigen operatively linked to the AAV shell polypeptide of claim 1 or claim 11 is administered to a subject in need of vaccination.

42. The use of claim 41, wherein the AAV shell polypeptide exhibits a lower seropositivity rate than the AAV2 shell polypeptide or the AAV shell polypeptide exhibits approximately the same or lower seropositivity rate as the AAV8 shell polypeptide.

43. The use of claim 41, wherein the AAV shell polypeptide is neutralized by human serum to a lesser extent than the AAV2 shell polypeptide, and the AAV shell polypeptide is neutralized by human serum to a similar or lesser extent than the AAV8 shell polypeptide.

44. Use of AAV particles for delivery of transgenic organisms, said AAV particles comprising a shell polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 and 17 or an amino acid sequence having at least 95% sequence identity with an amino acid sequence selected from the group consisting of: SEQ ID NO: 19, 20, 21, 22, 23, 24, 25 and 26.

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