-
Journal of Virology Sep 2021Recombinant adeno-associated viruses (rAAVs) are one of the most commonly used vectors for a variety of gene therapy applications. In the last 2 decades, research...
Recombinant adeno-associated viruses (rAAVs) are one of the most commonly used vectors for a variety of gene therapy applications. In the last 2 decades, research focused primarily on the characterization and isolation of new , genes resulting in hundreds of natural and engineered AAV capsid variants, while the gene, the other major AAV open reading frame, has been less studied. This is due to the fact that the gene from AAV serotype 2 (AAV2) enables the single-stranded DNA packaging of recombinant genomes into most AAV serotype and engineered capsids. However, a major by-product of all vector productions is empty AAV capsids, lacking the encapsidated vector genome, especially for non-AAV2 vectors. Despite the packaging process being considered the rate-limiting step for rAAV production, none of the genes from the other AAV serotypes have been characterized for their packaging efficiency. Thus, in this study AAV2 was replaced with the gene of a select number of AAV serotypes. However, this led to a lowering of capsid protein expression, relative to the standard AAV2- system. In further experiments the 3' end of the AAV2 gene was reintroduced to promote increased capsid expression and a series of chimeras between the different AAV Rep proteins were generated and characterized for their vector genome packaging ability. The utilization of these novel Rep hybrids increased the percentage of genome containing (full) capsids approximately 2- to -4-fold for all of the non-AAV2 serotypes tested. Thus, these Rep chimeras could revolutionize rAAV production. A major by-product of all adeno-associated virus (AAV) vector production systems are "empty" capsids, void of the desired therapeutic gene, and thus do not provide any curative benefit for the treatment of the targeted disease. In fact, empty capsids can potentially elicit additional immune responses gene therapies if not removed by additional purification steps. Thus, there is a need to increase the genome packaging efficiency and reduce the number of empty capsids from AAV biologics. The novel Rep hybrids from different AAV serotypes described in this study are capable of reducing the percentage of empty capsids in all tested AAV serotypes and improve overall yields of genome-containing AAV capsids at the same time. They can likely be integrated easily into existing AAV manufacturing protocols to optimize the production of the generated AAV gene therapy products.
Topics: Capsid; Capsid Proteins; DNA-Binding Proteins; Dependovirus; Genes, Viral; Genetic Vectors; HEK293 Cells; Humans; Recombinant Fusion Proteins; Viral Genome Packaging; Viral Proteins
PubMed: 34287038
DOI: 10.1128/JVI.00773-21 -
Nature Methods Jul 2023The development of transgenic mouse models that express genes of interest in specific cell types has transformed our understanding of basic biology and disease. However,...
The development of transgenic mouse models that express genes of interest in specific cell types has transformed our understanding of basic biology and disease. However, generating these models is time- and resource-intensive. Here we describe a model system, SELective Expression and Controlled Transduction In Vivo (SELECTIV), that enables efficient and specific expression of transgenes by coupling adeno-associated virus (AAV) vectors with Cre-inducible overexpression of the multi-serotype AAV receptor, AAVR. We demonstrate that transgenic AAVR overexpression greatly increases the efficiency of transduction of many diverse cell types, including muscle stem cells, which are normally refractory to AAV transduction. Superior specificity is achieved by combining Cre-mediated AAVR overexpression with whole-body knockout of endogenous Aavr, which is demonstrated in heart cardiomyocytes, liver hepatocytes and cholinergic neurons. The enhanced efficacy and exquisite specificity of SELECTIV has broad utility in development of new mouse model systems and expands the use of AAV for gene delivery in vivo.
Topics: Mice; Animals; Genetic Vectors; Gene Transfer Techniques; Mice, Transgenic; Genetic Therapy; Transgenes; Dependovirus; Transduction, Genetic
PubMed: 37291262
DOI: 10.1038/s41592-023-01896-x -
Frontiers in Immunology 2021Adeno-associated virus (AAV) vector-based gene therapy is currently the only gene therapy approved in the US and Europe. The recent tragic death of three children in a... (Review)
Review
Adeno-associated virus (AAV) vector-based gene therapy is currently the only gene therapy approved in the US and Europe. The recent tragic death of three children in a clinical trial to treat X-Linked Myotubular Myopathy by delivering myotubularin with an AAV8 vector notwithstanding, AAV remains a highly promising therapeutic gene delivery platform. But the successful use of AAV vectors to treat an increasing number of diseases also makes establishing protocols to determine therapeutically relevant titers of pre-existing anti-AAV antibodies and approaches to deplete those antibodies more urgent than ever. In this mini review, I will briefly discuss (i) our knowledge regarding the prevalence of anti-AAV antibodies, (ii) the challenges to measure those antibodies by methods that are most predictive of their influence on therapeutic efficacy of AAV gene transfer, and (iii) approaches to overcome the formidable hurdle that anti-AAV antibodies pose to the successful clinical use of AAV gene therapy.
Topics: Animals; Antibodies, Neutralizing; Antibodies, Viral; Biological Assay; Dependovirus; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Humans
PubMed: 33815421
DOI: 10.3389/fimmu.2021.658399 -
EMBO Molecular Medicine Apr 2021Gene therapy using recombinant adeno-associated virus (rAAV) vectors to treat blinding retinal dystrophies has become clinical reality. Therapeutically impactful...
Gene therapy using recombinant adeno-associated virus (rAAV) vectors to treat blinding retinal dystrophies has become clinical reality. Therapeutically impactful targeting of photoreceptors still relies on subretinal vector delivery, which detaches the retina and harbours substantial risks of collateral damage, often without achieving widespread photoreceptor transduction. Herein, we report the development of novel engineered rAAV vectors that enable efficient targeting of photoreceptors via less invasive intravitreal administration. A unique in vivo selection procedure was performed, where an AAV2-based peptide-display library was intravenously administered in mice, followed by isolation of vector DNA from target cells after only 24 h. This stringent selection yielded novel vectors, termed AAV2.GL and AAV2.NN, which mediate widespread and high-level retinal transduction after intravitreal injection in mice, dogs and non-human primates. Importantly, both vectors efficiently transduce photoreceptors in human retinal explant cultures. As proof-of-concept, intravitreal Cnga3 delivery using AAV2.GL lead to cone-specific expression of Cnga3 protein and rescued photopic cone responses in the Cnga3 mouse model of achromatopsia. These novel rAAV vectors expand the clinical applicability of gene therapy for blinding human retinal dystrophies.
Topics: Animals; Capsid; Color Vision Defects; Dependovirus; Dogs; Genetic Therapy; Genetic Vectors; Mice; Retina
PubMed: 33616280
DOI: 10.15252/emmm.202013392 -
Journal of Controlled Release :... Mar 2023In recent years, adeno-associated virus (AAV) has become the most important vector for central nervous system (CNS) gene therapy. AAV has already shown promising results... (Review)
Review
In recent years, adeno-associated virus (AAV) has become the most important vector for central nervous system (CNS) gene therapy. AAV has already shown promising results in the clinic, for several CNS diseases that cannot be treated with drugs, including neurodegenerative diseases, neuromuscular diseases, and lysosomal storage disorders. Currently, three of the four commercially available AAV-based drugs focus on neurological disorders, including Upstaza for aromatic l-amino acid decarboxylase deficiency, Luxturna for hereditary retinal dystrophy, and Zolgensma for spinal muscular atrophy. All these studies have provided paradigms for AAV-based therapeutic intervention platforms. AAV gene therapy, with its dual promise of targeting disease etiology and enabling 'long-term correction' of disease processes, has the advantages of immune privilege, high delivery efficiency, tissue specificity, and cell tropism in the CNS. Although AAV-based gene therapy has been shown to be effective in most CNS clinical trials, limitations have been observed in its clinical applications, which are often associated with side effects. In this review, we summarized the therapeutic progress, challenges, limitations, and solutions for AAV-based gene therapy in 14 types of CNS diseases. We focused on viral vector technologies, delivery routes, immunosuppression, and other relevant clinical factors. We also attempted to integrate several hurdles faced in clinical and preclinical studies with their solutions, to seek the best path forward for the application of AAV-based gene therapy in the context of CNS diseases. We hope that these thoughtful recommendations will contribute to the efficient translation of preclinical studies and wide application of clinical trials.
Topics: Humans; Central Nervous System Diseases; Central Nervous System; Genetic Therapy; Dependovirus; Immune Tolerance; Genetic Vectors
PubMed: 36736907
DOI: 10.1016/j.jconrel.2023.01.067 -
Signal Transduction and Targeted Therapy Apr 2024Adeno-associated virus (AAV) has emerged as a pivotal delivery tool in clinical gene therapy owing to its minimal pathogenicity and ability to establish long-term gene... (Review)
Review
Adeno-associated virus (AAV) has emerged as a pivotal delivery tool in clinical gene therapy owing to its minimal pathogenicity and ability to establish long-term gene expression in different tissues. Recombinant AAV (rAAV) has been engineered for enhanced specificity and developed as a tool for treating various diseases. However, as rAAV is being more widely used as a therapy, the increased demand has created challenges for the existing manufacturing methods. Seven rAAV-based gene therapy products have received regulatory approval, but there continue to be concerns about safely using high-dose viral therapies in humans, including immune responses and adverse effects such as genotoxicity, hepatotoxicity, thrombotic microangiopathy, and neurotoxicity. In this review, we explore AAV biology with an emphasis on current vector engineering strategies and manufacturing technologies. We discuss how rAAVs are being employed in ongoing clinical trials for ocular, neurological, metabolic, hematological, neuromuscular, and cardiovascular diseases as well as cancers. We outline immune responses triggered by rAAV, address associated side effects, and discuss strategies to mitigate these reactions. We hope that discussing recent advancements and current challenges in the field will be a helpful guide for researchers and clinicians navigating the ever-evolving landscape of rAAV-based gene therapy.
Topics: Humans; Dependovirus; Genetic Vectors; Genetic Therapy
PubMed: 38565561
DOI: 10.1038/s41392-024-01780-w -
Science Translational Medicine Mar 2022Prime editing is a highly versatile CRISPR-based genome editing technology that works without DNA double-strand break formation. Despite rapid technological advances, in...
Prime editing is a highly versatile CRISPR-based genome editing technology that works without DNA double-strand break formation. Despite rapid technological advances, in vivo application for the treatment of genetic diseases remains challenging. Here, we developed a size-reduced Cas9 prime editor (PE) lacking the RNaseH domain (PE2) and an intein-split construct (PE2 p.1153) for adeno-associated virus-mediated delivery into the liver. Editing efficiencies reached 15% at the locus and were further elevated to 58% by delivering unsplit PE2 via human adenoviral vector 5 (AdV). To provide proof of concept for correcting a genetic liver disease, we used the AdV approach for repairing the disease-causing mutation in a mouse model of phenylketonuria (PKU) via prime editing. Average correction efficiencies of 11.1% (up to 17.4%) in neonates led to therapeutic reduction of blood phenylalanine, without inducing detectable off-target mutations or prolonged liver inflammation. Although the current in vivo prime editing approach for PKU has limitations for clinical application due to the requirement of high vector doses (7 × 10 vg/kg) and the induction of immune responses to the vector and the PE, further development of the technology may lead to curative therapies for PKU and other genetic liver diseases.
Topics: Animals; Dependovirus; Gene Editing; Liver Diseases; Mice; Phenylketonurias
PubMed: 35294257
DOI: 10.1126/scitranslmed.abl9238 -
Human Gene Therapy Feb 2020Adeno-associated viral vectors have been successfully used in laboratory and clinical settings for efficient gene delivery. In these vectors, 96% of the adeno-associated...
Adeno-associated viral vectors have been successfully used in laboratory and clinical settings for efficient gene delivery. In these vectors, 96% of the adeno-associated virus (AAV) genome is replaced with a gene cassette of interest, leaving only the 145 bp inverted terminal repeat (ITR) sequences. These cis-elements, primarily from AAV serotype 2, are required for genome rescue, replication, packaging, and vector persistence. Previous work from our lab and others have demonstrated that the AAV ITR2 sequence has inherent transcriptional activity, which may confound intended transgene expression in therapeutic applications. Currently, AAV capsids are extensively study for vector contribution; however, a comprehensive analysis of ITR promoter activity of various AAV serotypes has not been described to date. Here, the transcriptional activity of AAV ITRs from different serotypes (1-4, 6, and 7) was compared in numerous cell lines and a mouse model. Under the conditions used here, all ITRs tested were capable of promoting transgene expression both and . However, we observed three classes of AAV ITR expression . Class I ITRs (AAV2 and 3) generated the highest level, whereas class II (AAV 4) had intermediate levels, and class III (AAV1 and 6) had the lowest levels. These expression levels were consistent across multiple cell lines. Only ITR7 demonstrated cell-type dependent transcriptional activity. , all classes had promoter activity. Next-generation sequencing revealed multiple transcriptional start sites that originated from the ITR sequence, with most arising from within the Rep binding element. The collective results demonstrate that the serotype ITR sequence may have multiple levels of influence on transgene expression cassettes independent of promoter selection.
Topics: Animals; Base Sequence; Cell Line; Dependovirus; Gene Expression; Gene Expression Regulation, Viral; Gene Transfer Techniques; Genes, Reporter; Genetic Engineering; Genetic Variation; Genetic Vectors; Humans; Mice; Nucleic Acid Conformation; Plasmids; Promoter Regions, Genetic; Serogroup; Terminal Repeat Sequences; Transcription Initiation Site; Transcriptional Activation; Transduction, Genetic; Transgenes
PubMed: 31914802
DOI: 10.1089/hum.2019.274 -
Nature Communications Oct 2023Large genes including several CRISPR-Cas modules like gene activators (CRISPRa) require dual adeno-associated viral (AAV) vectors for an efficient in vivo delivery and...
Large genes including several CRISPR-Cas modules like gene activators (CRISPRa) require dual adeno-associated viral (AAV) vectors for an efficient in vivo delivery and expression. Current dual AAV vector approaches have important limitations, e.g., low reconstitution efficiency, production of alien proteins, or low flexibility in split site selection. Here, we present a dual AAV vector technology based on reconstitution via mRNA trans-splicing (REVeRT). REVeRT is flexible in split site selection and can efficiently reconstitute different split genes in numerous in vitro models, in human organoids, and in vivo. Furthermore, REVeRT can functionally reconstitute a CRISPRa module targeting genes in various mouse tissues and organs in single or multiplexed approaches upon different routes of administration. Finally, REVeRT enabled the reconstitution of full-length ABCA4 after intravitreal injection in a mouse model of Stargardt disease. Due to its flexibility and efficiency REVeRT harbors great potential for basic research and clinical applications.
Topics: Humans; Animals; Mice; Trans-Splicing; Gene Editing; Genetic Therapy; Stargardt Disease; Genetic Vectors; Dependovirus; ATP-Binding Cassette Transporters
PubMed: 37852949
DOI: 10.1038/s41467-023-42386-0 -
Nature Biotechnology Jan 2022Gene therapy would benefit from a miniature CRISPR system that fits into the small adeno-associated virus (AAV) genome and has high cleavage activity and specificity in...
Gene therapy would benefit from a miniature CRISPR system that fits into the small adeno-associated virus (AAV) genome and has high cleavage activity and specificity in eukaryotic cells. One of the most compact CRISPR-associated nucleases yet discovered is the archaeal Un1Cas12f1. However, Un1Cas12f1 and its variants have very low activity in eukaryotic cells. In the present study, we redesigned the natural guide RNA of Un1Cas12f1 at five sites: the 5' terminus of the trans-activating CRISPR RNA (tracrRNA), the tracrRNA-crRNA complementary region, a penta(uridinylate) sequence, the 3' terminus of the crRNA and a disordered stem 2 region in the tracrRNA. These optimizations synergistically increased the average indel frequency by 867-fold. The optimized Un1Cas12f1 system enabled efficient, specific genome editing in human cells when delivered by plasmid vectors, PCR amplicons and AAV. As Un1Cas12f1 cleaves outside the protospacer, it can be used to create large deletions efficiently. The engineered Un1Cas12f1 system showed efficiency comparable to that of SpCas9 and specificity similar to that of AsCas12a.
Topics: CRISPR-Cas Systems; Dependovirus; Endonucleases; Gene Editing; Humans; RNA; RNA, Guide, CRISPR-Cas Systems
PubMed: 34475560
DOI: 10.1038/s41587-021-01009-z