Hybrid adeno-associated virus (AAV) particles and the methodology of assembly and uses thereof

Abstract

The present application relates to compositions and methodology for hybrid AAV vector-based pseudovirus with a protein coat derived for the Human Papillomavirus (HPV) or capsid proteins of other viruses. Using HPV as an example, the present inventors demonstrate that the HPV L1 and L2 proteins form a hybrid pseudovirus that packages an AAV genome-derived, large-sized, single-stranded DNA construct following DNA amplification in an engineered producer cell; the producer cell is engineered to expresses the AAV Rep gene, which amplifies the large-sized, single-stranded DNA for packaging into the pseudo-virion of HPV.

Description

[0001] Hybrid Adeno-Associated Virus (AAV) Particles and the Methodology of Assembly and Uses Thereof

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to U.S. Provisional Application No. 63 / 587,841, filed October 4, 2023, which application is incorporated by reference herein in its entirety.

[0004] FIELD

[0005] The present application relates to compositions and methodology for hybrid AAV vector-based pseudovirus with a protein coat derived for the Human Papillomavirus (HPV) or capsid proteins of other viruses including but not limited to norovirus, enterovirus, adenovirus, and rhinovirus.

[0006] INTRODUCTION

[0007] Virus particles are normally made up of two to three components - (i) A nucleic acid genome, either in the form of DNA or RNA; (ii) a protein coat, called capsid, that packages the viral genome inside the particle and protect the genome; (iii) some viruses also possess an outer lipid envelop that is derived from host cell membranes. Viruses with the lipid outer envelope are called “enveloped viruses” whereas those without the lipid membrane are called “non-enveloped viruses.” The packaging of viral genomes into viral capsid is a highly- regulated process. Different viruses package different genomes, and viruses can be classified into different groups based on the types of genomes that they package. For example, in the Baltimore Classification System, viruses are classified into seven groups based on the types of genomes that they specifically package (Koonin, Krupovic et al. 2021).

[0008] Group I: Double-stranded (ds) DNA viruses, which are the viruses that only encapsidate a dsDNA genome. This group of viruses includes viruses such as Herpes Simplex Virus (HSV) (Mcgeoch 1987) and Human Papillomavirus (HPV) (Yu, Majerciak et al. 2022).

[0009] Group II: single-stranded (ss) DNA viruses that only encapsidate a ssDNA genome. This group of viruses includes viruses such as Adeno-associated virus (AAV) (Wang, Tai et al. 2019).

[0010] Group III: double-stranded (ds) RNA viruses that only package a dsRNA genome. This group of viruses includes viruses such as rotaviruses (Estes and Cohen 1989) Group IV: positive-sense single-stranded (+ss) RNA viruses that only package a +ssRNA genome. The viral +ssRNA genome has the same polarity as cellular mRNA and can be translated directly. This group of viruses include viruses such as SARS-CoV and SARS-CoV-2 (V’kovski, Kratzel et al. 2020)

[0011] Group V: negative-sense single-stranded (-ss)RNA viruses that only package a - ssRNA genome. The viral -ssRNA genome has complementary polarity to cellular mRNA and cannot be translated directly. The -ssRNA genome needs to be transcribed to produce mRNA for translation. This group of viruses include viruses such as Influenza A (Bouvier and Palese 2008)

[0012] Group VI: RNA retroviruses: single-stranded RNA viruses that only package a positive-sense ssRNA genome that is later reverse-transcribed into a DNA intermediate in cells. The DNA genome is integrated into cellular genome for producing viral mRNA. This group of viruses include viruses such as Human Immunodeficiency Virus (HIV) (Barre- Sinoussi, Chermann et al. 1983).

[0013] Group VII: DNA retroviruses: double-stranded DNA viruses that only package a dsDNA genome that is revere-transcribed from RNA. This group of viruses include viruses such as Hepatitis B Virus (HBV) (Tsukuda and Watashi 2020).

[0014] The packaging of viral genomes into viral particles is normally a specific and highly- regulated process. It involves specific interactions of nucleic acids elements on viral genomes with specific domains of viral capsid proteins. For example, the packaging of the HIV RNA genome into virus particles involves specific interaction of the HIV capsid (Gag) with a RNA segment, called “Package signal (y), which is a stretch of ~ 150 to 250 bases near the 5’ end of the viral genomic RNA. The package signal is highly structured with several conserved stem-loops that interact with the zinc-knuckle (ZK) domains (with the consensus sequence: CX2CX4HX4C) of the HIV nuclear capsid (NC) (Mouhand, Pasi et al. 2020). The process mediates selective packaging of HIV genomic RNA into viral particles (Ding, Kharytonchyk et al. 2020).

[0015] The size of viral genomic DNA or RNA that can be packaged into viral particles varies from virus to virus, and is determined by the properties of viral capsids. For example, for Adeno-associated virus (AAV) genome packaging, the single-stranded genomic DNA size is normally limited to 4.7 kb (kilobase), and the packaged AAV genomes can never exceed 5.2 kb in length regardless of the capsid type (Wu, Yang et al. 2010). In addition, the nucleic acids conformation of viral genomes is also normally confined for individual viruses. For examples, adenoviruses only package a double-stranded, linear DNA genome of approximately 36 kb (Davison, Benko et al. 2003), whereas the human papillovmaviruses (HPV) only package a double-stranded, circular DNA genome of approximately 8 kb (Yu, Majerciak et al. 2022).

[0016] The genomic size and conformational constraint frequently pose a great challenge for using viral vectors for gene delivery. For example, for AAV vector, the total genomic size limit is 4.7 kb, and the AAV vector is normally not suitable for delivering therapeutic genes greater than 4 kb (Wu, Yang et al. 2010).

[0017] SUMMARY

[0018] The present application provides compositions and methodology for hybrid AAV vector-based pseudovirus with a protein coat derived for the Human Papillomavirus (HPV) or capsid proteins of other viruses.

[0019] HPV is a double-stranded DNA virus that packages a circular, double-stranded DNA genome. Traditional HPV pseudoviruses developed previously only package double-stranded, circular DNA vectors.

[0020] Using HPV as an example, the present inventors demonstrate that the HPV LI and L2 proteins form a hybrid pseudovirus that packages an AAV genome-derived, large-sized, single- stranded DNA construct following DNA amplification in an engineered producer cell; the producer cell is engineered to expresses the AAV Rep gene, which amplifies the largesized, single-stranded DNA for packaging into the pseudo-virion of HPV. HPV is a doublestranded DNA virus that packages a circular, double-stranded DNA genome. Traditional HPV pseudoviruses developed previously only package double-stranded, circular DNA vectors.

[0021] In no way limiting, the present application provides several advantages: (1) allows single- stranded, larger-than-AAV genome sized (4.7 kb), AAV genome-derived DNA constructs to be packaged into the pseudo-virion of HPV; these large sized (greater than 4.7 kb), single-stranded, AAV genome-derived DNA constructs are normally excluded from packaging into AAV particles. (2) allows single-stranded, linear DNA constructs to be packaged into the pseudo-virion of HPV.

[0022] An instant hybrid AAV vector-derived pseudoviruses produce robust gene expression in target cells and can be used for the delivery of large-sized therapeutic and reporter genes for disease treatment and diagnostic applications. An instant hybrid HPV-AAV pseudovirus (HP AV) can also be used for expressing reporter genes for anti-viral drug screening and antibody neutralization assays.

[0023] In one aspect, provided is a hybrid virus particle, comprising structural proteins LI and / or L2 from a papillomavirus species from papillomaviridae family, packaged together with a single-stranded, linear DNA genome derived from an AAV genome that is distinct from the papillomavirus species.

[0024] In one embodiment, the structural proteins LI and / or L2 come from HPV.

[0025] In one embodiment, the structural proteins LI and / or L2 form a particle that is assembled from one or more papillomavirus species and encapsulates the AAV genome- derived DNA genome.

[0026] In one embodiment, the AAV genome-derived DNA is derived from any AAV serotype. In a further embodiment, the AAV serotype is selected from AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, and AAVrhlO.

[0027] In another embodiment, the AAV genome is a self-complementary genome or two independently complementary AAV genomes derived from any AAV serotype. In a further embodiment, the AAV serotype is selected from AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, and AAVrhlO with mutant ITRs.

[0028] In another embodiment, each hybrid virus particle comprises structural proteins LI and / or L2 from a papillomavirus species from papillomaviridae family, packaged together with a single-stranded, linear DNA genome derived from an AAV genome that is distinct from the papillomavirus species.

[0029] In another aspect, provided is a method for assembling one or more hybrid virus particles, comprising:

[0030] (a) co-expressing papillomaviridae structural proteins LI and / or L2 with an AAV genome- derived DNA genome, in a mammalian cell line that expresses an AAV Rep gene and / or one or more helper genes; and

[0031] (b) harvesting one or more hybrid virus particles from cell supernatant or cell lysate.

[0032] In one embodiment, the papillomaviridae structural proteins LI and / or L2 are from a single papillomaviridae species or combinations of species. In a further embodiment, the one or more helper genes is selected from Adeno E2a and E4 genes. In one embodiment, the mammalian cell line is HEK293 or other mammalian cell line. In a further embodiment, the one or more helper genes is selected from Adeno E2a and E4 genes.

[0033] In one embodiment, the one or more helper genes is selected from Adeno E2a and E4 genes.

[0034] In another aspect, provided is a system and method for assembling the one or more hybrid virus particles in any of the preceding claims, wherein the hybrid particle assembly occurs in a producer cell that transiently or stably expresses the AAV Rep gene and / or the adenovirus helper genes including E2a and E4 genes, such that the producer cell allows the replication of the AAV genome-derived DNA construct and the encapsulation by the papillomaviridae structural proteins LI and / or L2, when LI and / or L2 genes are introduced into the producer cell.

[0035] In one embodiment, for the one or more hybrid virus particles, the particle's design effectively facilitates the delivery of an AAV DNA genome to target tissues and cells, thereby serving as a means for gene therapy, cancer treatment or prevention, and vaccines specifically targeting papillomaviruses.

[0036] In one embodiment, for the one or more hybrid virus particles, the AAV-genome based DNA genome is about 16 KB or more in length, with one or more ORFs.

[0037] In one embodiment, for the one or more hybrid virus particles, any gene of interest can be placed in the 16 KB AAV genome-derived genome and used for research, therapeutics, and diagnostic applications.

[0038] In one embodiment, for the one or more hybrid virus particles, the 16 KB AAV genome-derived genome comprises a gene of interest, wherein the gene of interest can encode a protein, a missing or malfunctioning protein, a therapeutic RNA, or other genetic elements to address a target disease or condition.

[0039] In one embodiment, for the one or more hybrid virus particles, the structural proteins are from distinct papillomavirus species or combinations of species within the papillomavirus family.

[0040] In one embodiment, for the one or more hybrid virus particles, the structural proteins are from non-enveloped viruses. In one embodiment, for the one or more hybrid virus particles, the structural proteins are selected from capsid proteins of norovirus, enterovirus, adenovirus, and rhinovirus.

[0041] In one embodiment, for the one or more hybrid virus particles, the particles are used for modeling norovirus and other papillomavirus infections, quantifying neutralizing antibodies, evaluating viral entry inhibiting drugs, and dissecting cellular factors crucial to viral replication.

[0042] In one embodiment, for the one or more hybrid virus particles, the particles are used for delivering an AAV-genome derived DNA genome to target tissues and cells, rendering them effective for gene therapy, cancer treatment or prevention, and vaccines tailored to HPV or other papillomaviruses.

[0043] In another embodiment, the HPV L1 / L2 capsid proteins are used to package other single- stranded DNA viral genomes including but not limited to those from parvovirus, densovirus, circovirus, nanovirus, geminivirus, and CRESS (Circular Rep-Encoding Single- Stranded) DNA viruses, using an instant hybrid virus particle, comprising structural proteins LI and / or L2 from a papillomavirus species from papillomaviridae family, packaged together with a single-stranded, linear DNA genome derived from an AAV genome that is distinct from the papillomavirus species..

[0044] In one embodiment, the producer cells are engineered, either transiently or stably, to express viral replicases to replicate viral linear genomes for packaging into L1 / L2 capsid using an instant hybrid virus particle.

[0045] In another aspect, provided is a any product, composition, kit, assay, methodology, system or use, as disclosed herein.

[0046] BRIEF DESCRIPTION OF THE FIGURES

[0047] Figure 1: Illustration of the HP AV particle and its genome.

[0048] (A) Illustration of AAV, HPV, and the hybrid HP AV particles and their genomes. The HP AV pseudovirus particle consists of the structural proteins (LI and L2) from HPV, and an AAV genome-derived single-stranded, linear DNA genome. (B) AAV-derived HP AV pseudovirus genome design with the expanded capacity to 16KB length between the ITRs. (C) Self- complementary AAV genome (scAAV) derived HP AV pseudovirus genome vector design with ITR and mutant ITRAtrs. Independently-complementary AAV genome (icAAV) derived HP AV pseudovirus genome vectors designed to package two complementary single-stranded linear genomes into HP AV.

[0049] Figure 2: Illustration for the production procedure of the HP AV particles.

[0050] (A) HEK293T cells were co-transfected with the plasmid vectors encoding the individual structural components of papillomaviruses (including LI and L2), an expression vector with Helper genes from Adenovirus, a plasmid with the AAV Rep gene, and a plasmid with the AAV-derived HP AV genome. Supernatants and cell lysates with the HP AV particles are harvested from cell supernatant or lysate 48-72hours post co-transfection, and can be purified and used to infect target cells. (B) an expanded form of the HP AV particles shown above in (A).

[0051] Figure 3: Tracking HPV 16 HP AV particle infection with a GFP reporter.

[0052] HEK293T cells were infected with HP AV particle with the HPV 16 LI and L2 structural proteins and an AAV genome with a GFP reporter. The GFP signal was observed by fluorescent microscopy 48 hours post infection and compared to a mock infection.

[0053] Figure 4: HPV 16 HPAV particle infection quantified by flow cytometry

[0054] Quantitative data was collected on the HPAV GFP particles with the HPV 16 shell using flow cytometry. (A) Mock infected HEK293T cells. (B) Infection of HEK293T cells with HPAV particles produced in cells expressing the AD Helper and the AAV Rep genes. The GFP signal was quantified by flow cytometry.

[0055] Figure 5: HPAV Pseudovirus Transduction Determined by Dosage and HPV Shell Serotype.

[0056] (A) Quantitative luciferase expression from the serial diluted HPAV pseudovirus with HPV 16 shell. (B) Luciferase quantification of the HPAV pseudoviruses infectivity with HPV shells from multiple serotypes. (C) GFP quantification of the HPAV pseudovirus transduction assembled with different HPV serotype shells.

[0057] Figure 6: HPAV Pseudovirus Quantification of Neutralizing Antibodies.

[0058] (A) A neutralization assay for a HP VI 6 neutralizing antibody was performed with HPAV pseudovirus and the infectivity was measured by GFP. (B) An IC50 curve and calculation determined using the percent infection and the loglO concentration of the antibody. Figure 7: HP AV Pseudovirus Quantification of Vaccine Induced Neutralizing Antibodies

[0059] (A) A quantitative neutralization assay for a serum donor against the HP AV pseudovirus for HPV 16. (B) HP AV pseudovirus neutralization assays for HPV 16 and HPV 31 for a second donor’s serum after vaccination with nine valent vaccine (C) HP AV pseudovirus neutralization assays for HPV 16 and HPV 31 for a third donor’s serum after vaccination with quadrivalent vaccine.

[0060] Figure 8: Illustration and Demonstration of HP AV Pseudovirus Transduction of an AAV Derived Genome Greater Than 4.7 kb

[0061] (A) An illustration of a ssAAV derived genome with two ORFs that has a total size of 6.2 kb.

[0062] (B) A GFP microscope image of HEK293T cells transduced by the HP AV pseudovirus with the 6.2 kb genome. (C) Quantification of the of HEK293T cells transduced by the HP AV pseudovirus with the 6.2 kb expressing firefly luciferase.

[0063] DETAILED DESCRIPTION

[0064] A. Adeno-associated viruses (AAVs)

[0065] Adeno-associated viruses (AAVs) are non-enveloped, singled-stranded DNA viruses with a single-stranded DNA genome size of 4.7 kb. AAVs have emerged as one of the safest and most effective vehicles for delivering genes into target cells for gene therapy. This approach has garnered significant attention due to its potential to provide long-term transgene expression and offer curative treatments for various diseases caused by genetic mutations.

[0066] AAV’s single-stranded DNA genome is flanked by two Inverted Terminal Repeats (ITRs). The genome can be modified to express transgenes (reporter genes or therapeutic genes) (Fig. 1A).

[0067] The AAV transgene expression cassette is designed to replace the AAV genome’s open reading frame (ORF) between the viral ITRs. The cassettes contain a promoter, gene of interest, terminator, regulatory elements and optional enhancers. A promoter is chosen to regulate gene expression. It can be a strong, ubiquitous promoter or a tissue-specific promoter, depending on the desired level and specificity of gene expression. Downstream of the promoter is the therapeutic or reporter gene inserted into the cassette. This gene can encode a protein, a missing or malfunctioning protein, a therapeutic RNA, or other genetic elements to address the target disease. Reporter genes like luciferase or green fluorescent protein (GFP) can be used for quantifying viral entry and transduction efficiencies. A terminator sequence is included to signal the end of gene transcription. Additional elements, such as enhancers, introns, polyadenylation sequences, and codon optimization, may be added to fine-tune transgene expression.

[0068] AAV vectors are assembled by first selecting an appropriate AAV capsid of certain serotype or variant based on the target tissue or cell type. Different AAV serotypes have distinct tissue tropisms. AAV particles are then assembled by co-transfecting plasmids encoding the selected capsid, a modified AAV genome of interest, adenovirus helper proteins, and the AAV Replicase. Particles assembled in producer cells can be purified and used to transduce target cells for transgene delivery. AAVs have been identified as ideal vectors for gene therapy due to their safety profile, versatility, and ability to drive long-term transgene expression in both dividing and non-dividing cells. AAV-mediated gene therapy is still in the early stages of clinical development, with only two commercialized products and most trials in the early phases. However, the field is rapidly expanding, driven by the urgent need for treatments for rare diseases and the increasing capacity to produce clinical-grade AAVs. The COVID-19 pandemic has further accelerated AAV production infrastructure development. For example, haemophilia A is one of the most targeted diseases, with numerous trials in late-phase development.

[0069] Despite the promise of AAV gene therapy, several challenges have hindered its application including limited gene packaging capacity, tissue tropism, lack of a determined dosing regime, and immunogenicity.

[0070] Traditional AAV vectors undergo several steps for transduction in target cells, including second-strand synthesis, which is often a rate-limiting factor in efficient transgene expression. This step is essential for converting the single-stranded AAV (ssAAV) genome into a double-stranded DNA (dsDNA) in target cells, but its inefficiency can lead to delayed or reduced transgene expression and even the loss of ssAAV genomes before conversion.

[0071] To overcome this bottleneck, a self-complementary AAV has been contemplated and engineered (scAAV) (Fig 1C). These vectors bypass second-strand synthesis in target cells by packaging a single-stranded genome that can self-anneal into a double-stranded form upon releasing the genome into the target cells. The modified AAV genome in scAAV vectors lacks a terminal resolution site (trs) in one of its inverted terminal repeats (ITRs), which allows for the packaging of a single-stranded DNA molecule that quickly self-complements into dsDNA after uncoating in target cells. By skipping the second-strand synthesis step, scAAV vectors can achieve up to 5- to 140-fold higher transgene expression compared to ssAAV vectors. This results in rapid onset of gene expression across various tissues, including the liver, muscle, brain, and central nervous system (CNS).

[0072] The scAAV vectors have demonstrated superior performance in delivering CRISPR- Cas9 components, achieving significant improvements in gene editing efficiency at lower doses. Due to their heightened transduction efficiency, scAAV vectors require significantly lower doses to achieve therapeutic effects, potentially reducing the risk of immune responses and enhancing overall safety. However, the self-complementary nature of scAAV limits its genome size to approximately 2.4-2.7 kb, which is smaller than the packaging capacity of conventional ssAAV vectors (~4 kb). This restricts the types of genes or therapeutic sequences that can be delivered.

[0073] There are numerous experiment approaches to increase the size of the genomes delivered by AAV. For example, a split vector approach can be used by splitting the gene into two pieces. Each piece of the gene is then independently packaged into an AAV (particle A and particle B). When a cell is co-infected with both A and B AVV particles, the fragments reassembled and produce the full-length gene. Nevertheless, such approach requires high multiplicity of infection (m.o.i) to reach co-infection of the same target cell with A and B, limiting practical application in cases when high moi infection is not achievable.

[0074] B. Human Papillomavirus (HPVs) and HPV Pseudoviruses

[0075] All Human Papillomavirus (HPVs) are non-enveloped double stranded DNA viruses. Their genomes are circular and approximately 8 kilobase pairs in size (Fig. 1A). HPV infection is one of the most common sexually transmitted diseases globally. It is strongly associated with the development of various cancers, including cervical cancer, head and neck squamous cell carcinoma (HNSCC), and anal cancer. Over 200 different types of HPV have been identified, each with distinct characteristics and clinical implications.

[0076] HPV infections are primarily transmitted through skin-to-skin or skin-to-mucosa contact. Some HPV types mainly infect cutaneous (skin) tissues, leading to the development of warts, while others primarily target mucosal tissues, such as those in the cervical and oral tracts. Depending on their oncogenic potential, mucosal HPV types are categorized as high- risk or low-risk. High-risk HPV types, like HPV16, 18, 31, and 33, have the potential to cause cancer, while low-risk types, like HPV6 and 11, typically lead to benign warts. Several HPV vaccines have been developed to provide protection against specific HPV types and have been shown to be effective in reducing the risk of HPV-related infections and cancers. Several HPV vaccines have been developed and licensed for use, including Cervarix, Gardasil, and Gardasil 9. These vaccines are based on virus-like particles (VLPs) of the major capsid protein LI of HPV. VLPs are non-infectious and non-oncogenic, making them a safe basis for vaccines. The vaccines differ in the HPV types they target and the adjuvants used.

[0077] • Cervarix protects against HP VI 6 and 18, the two most common oncogenic genotypes responsible for approximately 70% of cervical cancers.

[0078] • Gardasil targets HPV6, 11, 16, and 18, offering protection not only against cervical cancers but also genital warts.

[0079] • Gardasil 9 covers a broader spectrum, protecting against HPV6, 11, 16, 18, 31, 33, 45, 53, and 58, potentially preventing around 90% of cervical cancers.

[0080] The introduction of HPV vaccines has had a significant impact on reducing the incidence of HPV-related diseases. A modeling analysis suggests that a combination of intensive HPV vaccination and regular screening could lead to a 97% reduction in cervical cancer cases by 2100.

[0081] Current HPV vaccines are prophylactic, meaning they prevent new infections but do not treat pre-existing HPV infections or related conditions. Researchers are actively working on therapeutic vaccines that trigger a cellular immune response to treat established infections and malignancies. These therapeutic vaccines come in various forms, including bacterial- vector, viral-vector, peptide, nucleic acid, and cell-based vaccines. Despite ongoing efforts, no therapeutic vaccine has yet provided a complete and irreversible regression of HPV- associated cancers. However, combination therapies, such as vaccines combined with immune checkpoint inhibitors, are showing promise in suppressing tumor growth and enhancing immune responses.

[0082] The capacity of HPV capsids to package a double-stranded 8kb circular DNA has been utilized to produce HPV pseudoviruses that encapsulate a reporter circular plasmid DNA as a surrogate for native HPV circular ds DNA genome (Kawana, Yoshikawa et al. 1998, Touze and Coursaget 1998, Shi, Liu et al. 2001). The assembly and production of these traditional HPV pseudoviruses rely on the use of an engineered HEK293TT producer cell, which overexpresses the SV40 virus large T antigen. HEK293TT cell can specifically amplify circular, double-stranded DNA vectors carrying the SV40 origin of DNA replication. During HPV pseudovirus production in HEK293TT cell, reporter genes have to be cloned into a circular, double-stranded DNA vector carrying the SV40 origin of replication to be replicated and packaged by the HPV LI protein to form pseudoviruses. The pseudoviruses produced from this method often require concentrating by ultracentrifugation in order to get significant titers for downstream applications, thus greatly limiting their large-scale production and use in vaccine and therapeutic development. In addition, for HPV pseudoviruses, the L1 / L2 capsid does not effectively package large double-stranded, circular DNA great than 8 kb in size, and does not package RNA molecules.

[0083] C. AAV pseudovirus technology: HPV pseudotype AAV particles (HP AV)

[0084] The present inventors developed new pseudovirus technology for AAV. This new HPV Ll / L2-psuedotyped AAV particle, termed “HP AV particle,” represents a significant advancement in AAV and HPV research and gene therapy fields.

[0085] In one embodiment, the particles comprise an AAV transgene cassette packaged into an HP AV pseudo-virion assembled from the LI and / or L2 capsid proteins of HPV. The traditional AAV capsid proteins can only package the AAV genome which is a singlestranded DNA limited to 4.7 kilobases, reducing its overall ability to carry larger transgenes. The hybrid HP AV particle utilizes the LI capsid and would have a carrying capacity closer to that of the HPV genome, around 8 kilobases, double-stranded circular DNA.

[0086] The present application allows HPV pseudo-virions to package single-stranded, linear DNA with a size of approximately 16 kb (molecular weight equivalent to 8 kb doublestranded DNA). In addition to ssAAV genome, the hybrid HP AV pseudovirus can package a single-stranded, self-complementary AAV genome (scAAV) reaching about 8kb or more in length. This is nearly 2 to 4 times the gene carrying capacity of the traditional ssAAV and scAAV vectors, opening the possibility to have larger gene cassettes, including the CRISPR- Cas system with guide RNA on a single vector. The instant AAV genomes of the present application can now include multiple ORFs for TALEN gene editing and others.

[0087] In one embodiment, instant hybrid HP AV particles are assembled in a unique assembly system, in which a specially engineered HEK293 producer cell is co-transfected by vectors encoding the HPV structural proteins and the AAV replicase, and by the AAV- genome derived expression vectors. To allow the HPV capsid to package the AAV-derived, single-stranded, linear DNA genome, HEK293 producer cell is engineered to express the AAV Rep gene, either transient or stably. In addition, the producer cell is also engineered to express Adenovirus-Helper genes (Adeno E2a and E4 genes) to allow the AAV Rep to replicate the single-stranded, linear DNA to high-levels. The actions of the AAV Rep and helper genes in the HEK293 producer cell allow the replication of the AAV-derived, singlestranded, linear DNA genome which is then packaged into the HP AV particles by the HPV LI and / or L2 proteins.

[0088] Notably, the incorporation of the AAV-derived DNA genomes into hybrid AAV pseudovirus extends to the capsids of other viruses including but not limited to norovirus, enterovirus, and rhinovirus. The application and utility of the present application also extend beyond the current HPV and AAV technologies. In one embodiment, the present pseudovirus system will have functionality to grant further capabilities for HPV therapeutic vaccine development while also expanding the AAV gene delivery field by increasing tissue tropism and increasing the gene packaging capacity.

[0089] In addition, an instant HPV L1 / L2 capsid can also be used to package other singlestranded DNA viral genomes such as those from parvovirus, densovirus, circovirus, nanovirus, geminivirus, and CRESS (Circular Rep-Encoding Single-Stranded) DNA viruses, using a similar design as described above. The producer cells can be engineered, either transiently or stably, to express viral replicases to replicate single-stranded viral genomes with the proper sizes (molecular weight approximately equivalent to that of 8 kb doublestranded DNA) for packaging into the L1 / L2 capsid of the hybrid pseudoviruses.

[0090] EXAMPLES

[0091] The below examples provide illustrative and non-limiting disclosure.

[0092] EXAMPLE 1: HYBRID HP AV PSEUDOVIRUS PARTICLE COMPOSITION AND GENOME STRUCTURE.

[0093] The present application provides compositions and methodology for making and using a hybrid HP AV pseudovirus parti cle(s). Fig. 1 A illustrates the differences between AAV particle, HPV particle, and the new hybrid HP AV pseudovirus particle in their compositions and genome structures.

[0094] The AAV particle is composed of the capsid protein that packages a 4.7 kb linear, single- stranded DNA genome. The HPV virus particle is comprised of LI and L2 structural proteins that packages an 8kb circular double stranded genome. The HP AV particles are comprised of the viral structural proteins of HPV including the primary structural protein LI and the minor capsid protein L2. The LI protein can assemble into a particle once it is expressed in a producer cell line like HEK293. The L2 increases particle stability but is not required for particle formation. Once the particle starts to assemble, it can encapsulate a single- stranded, linear DNA genome up to 16 kb in length in the presence of ssDNA replication mediated by viral replicases.

[0095] For making and using the present HP AV particles, the AAV genome-derived, singlestranded, linear DNA genome that is encapsulated by the LI or L1 / L2 HPV capsid assembly. The DNA consists of the AAV ITRs and can carry multiple ORFs with several different promoters that can be tissue or viral derived. The ORFs can range in size to 16 Kb or more, and encode full length proteins such as the proteins involved with the CRISPR-Cas system and promoters to generate the guide RNAs. (C) diagram of an HP AV genome with the linear, single- stranded, self-complementary (LSSC) AAV-derived genome or linear, single-stranded, independently-complementary (LSIC) AAV-derived genomes. In LSSC, one of the ITRs have been replaced with the mutant ITR with the trs removed. The LSSC ORFs can range in size to around 4 Kb or more in length, whereas in LSIC, the ORFs can range in size to around 8 Kb or more in length.

[0096] EXAMPLE 2: ASSEMBLING HP AV PARTICLES.

[0097] The present application provides compositions and methodology for making and using a hybrid HP AV pseudovirus particle(s).

[0098] While in no way limiting, Fig. 2 discloses an example method employed to assemble HP AV particles. Vectors expressing the essential shell proteins of the HPV family, LI and L2 are co-transfected with the vectors carrying the AAV genome-derived vector (as described in Fig. 1), the vector for AAV helper genes from the adenovirus (AD helper), and the vector for AAV replicase (AAV Rep) gene. These co-transfections occur within human cells, such as HEK293 cells (producer cell). After 48 hours, virion particles are harvested form the cell lysate and further purification is achievable through a series of purification techniques, including sucrose gradient centrifugation. The purified particles are primed for utilization in pseudovirus neutralization assays, gene transduction or immunization. The producer cell is also engineered to stably express the AAV Rep gene and the AAV helper genes.

[0099] EXAMPLE 3: HP AV PARTICLES TO INFECT TARGET CELLS AND EXPRESS A GENE OF INTEREST (GFP). The present application provides compositions and methodology for making and using a hybrid HP AV pseudovirus particle(s), such as for infecting target cells, as determined by GFP reported expression.

[0100] As shown in Fig. 3, the present HP AV particles can infect target cells and express a gene of interest, such as a GFP reporter gene. HP AV particles were assembled as described above and subsequently employed to infect target HEK293 cells. Within 48 hours of infection, the vibrant expression of GFP was evident, underscoring the proficiency of HP AV particles to initiate infection and trigger the expression of the reporter gene within the target cells.

[0101] EXAMPLE 4: QUANTIFICATION OF THE INFECTIVITY OF HP AV PARTICLES.

[0102] The present application provides compositions and methodology for making and using a hybrid HP AV pseudovirus particle(s), such as for infecting target cells, as quantified by flow cytometry.

[0103] As exemplified in Fig. 4, quantification of the infectivity of HP AV particles can be determined by methods known in the art, such as by flow cytometry. The mock infected HEK293 cells do not show any GFP signal. In contrast, the HEK293 cells infected with HP AV particles produced in producer cells with the AD Helper and the AAV Rep genes show GFP signal and the particles were able to infect over 20% of the cells. The flow cytometry data here shows the infectious titer of the HP AV particles is greater than 5xl04infectious units per milliliter and in this round of infection over 20% of the target cells were transduced.

[0104] EXAMPLE 5: HP AV PSEUDOVIRUS EXPRESSES A GENE OF INTEREST IN A DOSAGE DEPENDENT MANNER.

[0105] The present application provides compositions and methodology for making and using a hybrid HP AV pseudovirus particle(s), such as for expressing a gene of interest in target cells.

[0106] In this regard, Fig.5 demonstrates the HP AV pseudovirus expresses the gene of interest in a dosage dependent manner and the LI and L2 structural proteins from various HPV serotypes are infectious when quantified by luciferase or GFP. As described above. HP AV pseudovirus was assembled with the HPV 16 LI and L2, serial diluted and the infectivity was quantified by luciferase, demonstrating gene expression was dependent on the dosage of the pseudovirus. As disclosed above, HP AV pseudoviruses were assembled with multiple HPV serotype shell proteins including, HPV-6, HPV-11,HPV-16, HPV-31, HPV-33, and HPV-45 and a ssAAV genome expressing luciferase. The pseudovirus infectivity on HEK293 cells show that the multiple serotypes are capable of infecting cells.

[0107] These results demonstrate the HP AV pseudoviruses with different HPV shells have differing transduction efficiencies, quantified by GFP expression in HEK293 cells.

[0108] EXAMPLE 6: HP AV PARTICLES FOR QUANTIFYING NEUTRALIZING ANTIBODIES FOR HPV.

[0109] The present application provides compositions and methodology for making and using a hybrid HP AV pseudovirus parti cle(s), such as the use of HP AV particles to quantify neutralizing antibodies for HPV.

[0110] The HP AV particles were assembled with the LI and L2 from HPV 16 and a ssAAV genome for GFP and were used to infect HEK293 cells in the presence of a HPV neutralizing antibody.

[0111] As shown in Fig.6, HP AV pseudovirus infectivity decreased with a higher concentration of neutralizing antibody. The plot illustrates the calculated IC50 of the HPV 16 neutralizing antibody and the antibody IC50 was determined to be 25.61 ng / mL.

[0112] EXAMPLE 7: HP AV PSEUDOVIRUS CAN MEASURE THE NEUTRALIZING ACTIVITY OF ANTIBODIES FROM VACCINATED HUMAN SERUM AND QUANTIFIES HPV SEROTYPE SPECIFIC RESPONSES.

[0113] The present application provides compositions and methodology for making and using a hybrid HP AV pseudovirus particle(s), such as for measuring the neutralizing activity of antibodies from vaccinated human serum and quantifying HPV serotype specific responses.

[0114] As shown in Fig. 7, HP AV pseudovirus can measure the neutralizing activity of antibodies from vaccinated human serum and quantify HPV serotype specific responses. A human donor serum was serial diluted, and the HP AV pseudovirus was used to measure the donor’s antibody neutralization IC50 dose to neutralize HPV16. A serum neutralization assay was performed with the HP AV pseudovirus and the serum from a second donor who was vaccinated with 9 valent vaccine (including HPV 31). The HP AV pseudovirus assay determined the IC50 for the HPV 16 of 1 : 563.7 and the HPV 31 of 1 : 1689.7, indicating a protective vaccine response for both HPV 16 and HPV 31.

[0115] A serum neutralization assay was performed with the HP AV pseudovirus, and serum from a third donor who was vaccinated with quadrivalent vaccine (not including HPV 31). The HP AV pseudovirus assay determined the IC50 for the HPV 16 of 1 : 1046 and the HPV 31 of 1 :28.7. indicating a protective response only to HPV 16 and no protective response to HPV 31.

[0116] These results demonstrate that the HP AV pseudovirus can determine specific vaccine responses.

[0117] EXAMPLE 8: ssAAV GENOME WITH TWO ORFS PACKAGED BY THE HPAV PARTICLE AND ITS INFECTIVITY IN HEK293 CELLS

[0118] The present application provides compositions and methodology for making and using a hybrid HPAV pseudovirus particle(s), such as a HPV Ll / L2-psuedotyped AAV particle (HPAV), comprising a large ssAAV genome reaching about 8kb or more in length, with one or more open reading frames (ORFs) and double-stranded circular DNA.

[0119] As shown in Figure 8, the ssAAV genome contains two ORFS, the first is a CMV promoter that expresses a firefly luciferase gene and eGFP from an IRES. The second expresses a puromycin selection marker from an SV40 promoter followed by a shared polyA signal. In total the genome is 6.2 kb in length.

[0120] An HPAV particle assembled with the HPV 16 LI and L2 and the 6.2 kb ssAAV genome is able to express GFP in HEK293 cells infected by the pseudovirus.

[0121] Further, the HPAV 6.2kb genome can express the firefly luciferase gene in addition to the GFP gene in HEK293 cells. This demonstrates the capability of the HPAV pseudovirus to transduce cells with an AAV genome greater than 6 kb.

[0122] These HPAV particles hold immense potential, including but not limited to enabling the identification of novel host cell receptors, quantification of viral neutralization for antibodies and antiviral drugs, assessment of viral variant infectivity, targeted delivery of therapeutic genes to specific cell populations, and initiation of immune responses as a preventive measure.

[0123] These particles also greatly impact the AAV-based gene therapy industry by expanding the cell tropisms for AAV and the gene carrying capacity, opening the door for the first time for a single AAV vector to carry full length versions of the CRISPR-CAS system, chimeric antigen receptors (CARs), Zinc Finger proteins, Talons, GM-CSF, ABCD1 gene, Beta-globin (1 lpl5) HBB gene, Connexin 26 (13ql 1), hexosaminidase A (15q23), Dystrophin (Xq21), CFTR (7q31), HFE (6p21), LDL receptor (19pl3)and Huntington (4pl6) gene.

[0124] References:

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[0131] Koonin, E. V., M. Krupovic and V. I. Agol (2021). "The Baltimore Classification of Viruses 50 Years Later: How Does It Stand in the Light of Virus Evolution?" Microbiol Mol Biol Rev 85(3): e0005321.

[0132] Mcgeoch, D. J. (1987). "The genome of herpes simplex virus: structure, replication and evolution." Journal of Cell Science 1987(Supplement_7): 67-94.

[0133] Mouhand, A., M. Pasi, M. Catala, L. Zargarian, A. Belfetmi, P. Barraud, O. Mauffret and C. Tisne (2020). "Overview of the Nucleic-Acid Binding Properties of the HIV-1 Nucleocapsid Protein in Its Different Maturation States." Viruses 12(10).

[0134] Shi, W., J. Liu, Y. Huang and L. Qiao (2001). "Papillomavirus pseudovirus: a novel vaccine to induce mucosal and systemic cytotoxic T-lymphocyte responses." J Virol 75(21): 10139- 10148. Touze, A. and P. Coursaget (1998). "In vitro gene transfer using human papillomavirus-like particles." Nucleic Acids Res 26(5): 1317-1323.

[0135] Tsukuda, S. and K. Watashi (2020). "Hepatitis B virus biology and life cycle." Antiviral Research 182: 104925. V’kovski, P., A. Kratzel, S. Steiner, H. Stalder and V. Thiel (2020). "Coronavirus biology and replication: implications for SARS-CoV-2." Nature Reviews Microbiology.

[0136] Wang, D., P. W. L. Tai and G. Gao (2019). "Adeno-associated virus vector as a platform for gene therapy delivery." Nature Reviews Drug Discovery 18(5): 358-378.

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Claims

What is claimed is:

1. A hybrid virus particle, comprising structural proteins LI and / or L2 from a papillomavirus species from papillomaviridae family, packaged together with a singlestranded, linear DNA genome derived from an AAV genome that is distinct from the papillomavirus species.

2. The hybrid virus particle of claim 1, wherein the structural proteins LI and / or L2 come from HPV.

3. The hybrid virus particle of claim 1, wherein the structural proteins LI and / or L2 form a particle that is assembled from one or more papillomavirus species and encapsulates the AAV genome-derived DNA genome.

4. The hybrid virus particle of claim 1, wherein the AAV genome-derived DNA is derived from any AAV serotype.

5. The hybrid virus particle of claim 4, wherein the AAV serotype is selected from AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, and AAVrhlO6. The hybrid virus particle of claim 1, wherein the AAV genome is a self- complementary genome or two independently complementary AAV genomes derived from any AAV serotype.

7. The hybrid virus particle of claim 6, wherein the AAV serotype is selected from AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, and AAVrhlO with mutant ITRs.

8. One or more of the hybrid virus particles of any of the preceding claims, wherein each hybrid virus particle comprises structural proteins LI and / or L2 from a papillomavirusspecies from papillomaviridae family, packaged together with a single-stranded, linear DNA genome derived from an AAV genome that is distinct from the papillomavirus species.

9. A method for assembling one or more hybrid virus particles, comprising:(a) co-expressing papillomaviridae structural proteins LI and / or L2 with an AAV genome- derived DNA genome, in a mammalian cell line that expresses an AAV Rep gene and / or one or more helper genes; and(b) harvesting one or more hybrid virus particles from cell supernatant or cell lysate.

10. The method of claim 9, wherein the papillomaviridae structural proteins LI and / or L2 are from a single papillomaviridae species or combinations of species.

11. The method of claim 9, wherein the mammalian cell line is HEK293 or other mammalian cell line.

12. The method of claim 9 or 11, wherein the one or more helper genes is selected from Adeno E2a and E4 genes.

13. A system and method for assembling the one or more hybrid virus particles in any of the preceding claims, wherein the hybrid particle assembly occurs in a producer cell that transiently or stably expresses the AAV Rep gene and / or the adenovirus helper genes including E2a and E4 genes, such that the producer cell allows the replication of the AAV genome-derived DNA construct and the encapsulation by the papillomaviridae structural proteins LI and / or L2, when LI and / or L2 genes are introduced into the producer cell.

14. The one or more hybrid virus particles in any of the preceding claims, wherein the particle's design effectively facilitates the delivery of an AAV DNA genome to target tissues and cells, thereby serving as a means for gene therapy, cancer treatment or prevention, and vaccines specifically targeting papillomaviruses.

15. The one or more hybrid virus particles in any of the preceding claims, wherein the AAV-genome based DNA genome is about 16 KB or more in length, with one or more ORFs.

16. The one or more hybrid virus particles in any of the preceding claims, wherein any gene of interest can be placed in the 16 KB AAV genome-derived genome and used for research, therapeutics, and diagnostic applications.

17. The one or more hybrid virus particles in any of the preceding claims, wherein the 16 KB AAV genome-derived genome comprises a gene of interest, wherein the gene of interest can encode a protein, a missing or malfunctioning protein, a therapeutic RNA, or other genetic elements to address a target disease or condition.

18. The one or more hybrid virus particles in any of the preceding claims, wherein the structural proteins are from distinct papillomavirus species or combinations of species within the papillomavirus family.

19. The one or more hybrid virus particles in any of the preceding claims, wherein the structural proteins are from non-enveloped viruses.

20. The one or more hybrid virus particles of claim 19, wherein the structural proteins are selected from capsid proteins of norovirus, enterovirus, adenovirus, and rhinovirus.

21. The one or more hybrid virus particles of any of the preceding claims, wherein the particles are used for modeling norovirus and other papillomavirus infections, quantifying neutralizing antibodies, evaluating viral entry inhibiting drugs, and dissecting cellular factors crucial to viral replication.

22. The one or more hybrid virus particles of any of the preceding claims, wherein the particles are used for delivering an AAV-genome derived DNA genome to target tissues and cells, rendering them effective for gene therapy, cancer treatment or prevention, and vaccines tailored to HPV or other papillomaviruses.

23. The HPV L1 / L2 capsid proteins are used to package other single-stranded DNA viral genomes including but not limited to those from parvovirus, densovirus, circovirus, nanovirus, geminivirus, and CRESS (Circular Rep-Encoding Single-Stranded) DNA viruses, using similar design of claim 1. The producer cells are engineered, either transiently or stably, to express viral replicases to replicate viral linear genomes for packaging into L1 / L2 capsid using similar design of claim 1.

24. Any product, composition, kit, assay, methodology, system, or use disclosed herein.

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