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The AAPS Journal Jul 2023Interest and efforts to use recombinant adeno-associated viruses (AAV) as gene therapy delivery tools to treat disease have grown exponentially. However, gaps in... (Review)
Review
Interest and efforts to use recombinant adeno-associated viruses (AAV) as gene therapy delivery tools to treat disease have grown exponentially. However, gaps in understanding of the pharmacokinetics/pharmacodynamics (PK/PD) and disposition of this modality exist. This position paper comes from the Novel Modalities Working Group (WG), part of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ). The pan-industry WG effort focuses on the nonclinical PK and clinical pharmacology aspects of AAV gene therapy and related bioanalytical considerations.Traditional PK concepts are generally not applicable to AAV-based therapies due to the inherent complexity of a transgene-carrying viral vector, and the multiple steps and analytes involved in cell transduction and transgene-derived protein expression. Therefore, we explain PK concepts of biodistribution of AAV-based therapies and place key terminologies related to drug exposure and PD in the proper context. Factors affecting biodistribution are presented in detail, and guidelines are provided to design nonclinical studies to enable a stage-gated progression to Phase 1 testing. The nonclinical and clinical utility of transgene DNA, mRNA, and protein analytes are discussed with bioanalytical strategies to measure these analytes. The pros and cons of qPCR vs. ddPCR technologies for DNA/RNA measurement and qualitative vs. quantitative methods for transgene-derived protein are also presented. Last, best practices and recommendations for use of clinical and nonclinical data to project human dose and response are discussed. Together, the manuscript provides a holistic framework to discuss evolving concepts of PK/PD modeling, bioanalytical technologies, and clinical dose selection in gene therapy.
Topics: Humans; Dependovirus; Tissue Distribution; Genetic Therapy; Drug Development; Polymerase Chain Reaction
PubMed: 37523051
DOI: 10.1208/s12248-023-00842-1 -
European Journal of Pharmaceutics and... Sep 2023Gene therapies offer promising therapeutic alternatives for many disorders that currently lack efficient treatment options. Due to their chemical nature and... (Review)
Review
Gene therapies offer promising therapeutic alternatives for many disorders that currently lack efficient treatment options. Due to their chemical nature and physico-chemical properties, delivery of polynucleic acids into target cells and subcellular compartments remains a significant challenge. Adeno-associated viruses (AAV) have gained a lot of interest for the efficient delivery of therapeutic single-stranded DNA (ssDNA) genomes over the past decades. More than a hundred products have been tested in clinical settings and three products have received market authorization by the US FDA in recent years. A lot of effort is being made to generate potent recombinant AAV (rAAV) vectors that show favorable safety and immunogenicity profiles for either local or systemic administration. Manufacturing processes are gradually being optimized to deliver a consistently high product quality and to serve potential market needs beyond rare indications. In contrast to protein therapeutics, most rAAV products are still supplied as frozen liquids within rather simple formulation buffers to enable sufficient product shelf life, significantly hampering global distribution and access. In this review, we aim to outline the hurdles of rAAV drug product development and discuss critical formulation and composition aspects of rAAV products under clinical evaluation. Further, we highlight recent development efforts in order to achieve stable liquid or lyophilized products. This review therefore provides a comprehensive overview on current state-of-the-art rAAV formulations and can further serve as a map for rational formulation development activities in the future.
Topics: Dependovirus; Genetic Vectors; Genetic Therapy
PubMed: 37423416
DOI: 10.1016/j.ejpb.2023.07.002 -
Human Gene Therapy Oct 2021Adeno-associated virus (AAV) is currently the most popular gene delivery vector for gene therapy. However, variability in titration methods between different...
Adeno-associated virus (AAV) is currently the most popular gene delivery vector for gene therapy. However, variability in titration methods between different laboratories affects the reproducibility of experiments and evaluation of safety and efficacy in clinical trials. We describe an optimized protocol for AAV titration, including quantitative PCR (qPCR) standard preparation and quantitation and treatment of AAV samples before qPCR and droplet digital PCR (ddPCR) titration. During the protocol development, we observed that quantitation of the qPCR standard was dependent on its conformation and that A260-based quantitation overestimated the plasmid copy numbers, introducing significant error. Linearized, free inverted terminal repeat (free-ITR), and supercoiled standards were compared with enhanced green fluorescent protein (EGFP), SV40p(A), and AAV2-ITR qPCR assays and we found that using the AAV2-ITR assay together with either linearized or supercoiled standard led to overestimation of the titers, while EGFP and SV40p(A) assays were more accurate with the linearized standard. Finally, we compared extraction of AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9 genomes by heat denaturation, proteinase K treatment, and kit extraction. Kit extraction, which contained proteinase K treatment in denaturing buffer before spin-column purification, significantly increased the titers acquired for all the serotypes in both qPCR and ddPCR. These improvements resulted in an accurate quantitation of the ATCC reference standard and in a robust and reliable protocol for AAV titration.
Topics: Dependovirus; Genetic Therapy; Genetic Vectors; Reproducibility of Results; Terminal Repeat Sequences
PubMed: 33560161
DOI: 10.1089/hum.2020.318 -
Biotechnology Journal Jan 2021For adeno-associated virus (AAV)-based human gene therapy, challenges for the translation of promising research results to successful clinical development include... (Review)
Review
For adeno-associated virus (AAV)-based human gene therapy, challenges for the translation of promising research results to successful clinical development include optimization of vector design and manufacturing processes to ensure that vectors prepared for administration to human subjects have attributes consistent with safe and durable expression. This article briefly reviews quality control methods for routine testing and supplemental characterization of AAV vectors for investigational product development. The relationship of vector and manufacturing process design with product critical quality attributes is discussed.
Topics: Dependovirus; Genetic Therapy; Genetic Vectors; Humans; Quality Control
PubMed: 33146911
DOI: 10.1002/biot.202000022 -
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 -
Human Gene Therapy Apr 2022In the past two decades, adeno-associated virus (AAV) vector manufacturing has made remarkable advancements to meet large-scale production demands for preclinical and...
In the past two decades, adeno-associated virus (AAV) vector manufacturing has made remarkable advancements to meet large-scale production demands for preclinical and clinical trials. In addition, AAV vectors have been extensively studied for their safety and efficacy. In particular, the presence of empty AAV capsids and particles containing "inaccurate" vector genomes in preparations has been a subject of concern. Several methods exist to separate empty capsids from full particles; but thus far, no single technique can produce vectors that are free of empty or partial (non-unit length) capsids. Unfortunately, the exact genome compositions of full, intermediate, and empty capsids remain largely unknown. In this work, we used AAV-genome population sequencing to explore the compositions of DNase-resistant, encapsidated vector genomes produced by two common production pipelines: plasmid transfection in human embryonic kidney cells (pTx/HEK293) and baculovirus expression vectors in insect cells (rBV/Sf9). Intriguingly, our results show that vectors originating from the same construct design that were manufactured by the rBV/Sf9 system produced a higher degree of truncated and unresolved species than those generated by pTx/HEK293 production. We also demonstrate that empty particles purified by cesium chloride gradient ultracentrifugation are not truly empty but are instead packaged with genomes composed of a single truncated and/or unresolved inverted terminal repeat (ITR). Our data suggest that the frequency of these "mutated" ITRs correlates with the abundance of inaccurate genomes in all fractions. These surprising findings shed new light on vector efficacy, safety, and how clinical vectors should be quantified and evaluated.
Topics: Animals; Baculoviridae; Dependovirus; Genetic Vectors; HEK293 Cells; Humans; Insecta
PubMed: 35293222
DOI: 10.1089/hum.2022.050 -
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 -
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 -
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