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Gene Therapy May 2024In this study, we demonstrate the safety and utility of CRISPR-Cas9 gene editing technology for in vivo editing of proviral DNA in ART-treated, virally controlled simian...
In this study, we demonstrate the safety and utility of CRISPR-Cas9 gene editing technology for in vivo editing of proviral DNA in ART-treated, virally controlled simian immunodeficiency virus (SIV) infected rhesus macaques, an established model for HIV infection. EBT-001 is an AAV9-based vector delivering SaCas9 and dual guide RNAs designed to target multiple regions of the SIV genome: the viral LTRs, and the Gag gene. The results presented here demonstrate that a single IV inoculation of EBT-001 at each of 3 dose levels (1.4 × 10, 1.4 × 10 and 1.4 × 10 genome copies/kg) resulted in broad and functional biodistribution of AAV9-EBT-001 to known tissue reservoirs of SIV. No off-target effects or abnormal pathology were observed, and animals returned to their normal body weight after receiving EBT-001. Importantly, the macaques that received the 2 highest doses of EBT-001 showed improved absolute lymphocyte counts as compared to antiretroviral-treated controls. Taken together, these results demonstrate safety, biodistribution, and in vivo proviral DNA editing following IV administration of EBT-001, supporting the further development of CRISPR-based gene editing as a potential therapeutic approach for HIV in humans.
Topics: Animals; Simian Immunodeficiency Virus; Macaca mulatta; Gene Editing; CRISPR-Cas Systems; Simian Acquired Immunodeficiency Syndrome; Tissue Distribution; Genetic Vectors; Genetic Therapy; Dependovirus
PubMed: 37587230
DOI: 10.1038/s41434-023-00410-4 -
Molecular Therapy : the Journal of the... Mar 2024Directed evolution of natural AAV9 using peptide display libraries have been widely used in the search for an optimal recombinant AAV (rAAV) for transgene delivery...
Directed evolution of natural AAV9 using peptide display libraries have been widely used in the search for an optimal recombinant AAV (rAAV) for transgene delivery across the blood-brain barrier (BBB) to the CNS following intravenous ( IV) injection. In this study, we used a different approach by creating a shuffled rAAV capsid library based on parental AAV serotypes 1 through 12. Following selection in mice, 3 novel variants closely related to AAV1, AAV-BBB6, AAV-BBB28, and AAV-BBB31, emerged as top candidates. In direct comparisons with AAV9, our novel variants demonstrated an over 270-fold improvement in CNS transduction and exhibited a clear bias toward neuronal cells. Intriguingly, our AAV-BBB variants relied on the LY6A cellular receptor for CNS entry, similar to AAV9 peptide variants AAV-PHP.eB and AAV.CAP-B10, despite the different bioengineering methods used and parental backgrounds. The variants also showed reduced transduction of both mouse liver and human primary hepatocytes in vivo. To increase clinical translatability, we enhanced the immune escape properties of our new variants by introducing additional modifications based on rational design. Overall, our study highlights the potential of AAV1-like vectors for efficient CNS transduction with reduced liver tropism, offering promising prospects for CNS gene therapies.
Topics: Humans; Animals; Mice; Blood-Brain Barrier; Genetic Therapy; Capsid; Liver; Peptides; Dependovirus; Genetic Vectors; Transduction, Genetic
PubMed: 38297833
DOI: 10.1016/j.ymthe.2024.01.024 -
Molecular Therapy : the Journal of the... Dec 2023Adeno-associated virus (AAV) continues to be the gold standard vector for therapeutic gene delivery and has proven especially useful for treating ocular disease....
Intravitreal injection of a rationally designed AAV capsid library in non-human primate identifies variants with enhanced retinal transduction and neutralizing antibody evasion.
Adeno-associated virus (AAV) continues to be the gold standard vector for therapeutic gene delivery and has proven especially useful for treating ocular disease. Intravitreal injection (IVtI) is a promising delivery route because it increases accessibility of gene therapies to larger patient populations. However, data from clinical and non-human primate (NHP) studies utilizing currently available capsids indicate that anatomical barriers to AAV and pre-existing neutralizing antibodies can restrict gene expression to levels that are "sub-therapeutic" in a substantial proportion of patients. Here, we performed a combination of directed evolution in NHPs of an AAV2-based capsid library with simultaneous mutations across six surface-exposed variable regions and rational design to identify novel capsid variants with improved retinal transduction following IVtI. Following two rounds of screening in NHP, enriched variants were characterized in intravitreally injected mice and NHPs and shown to have increased transduction relative to AAV2. Lead capsid variant, P2-V1, demonstrated an increased ability to evade neutralizing antibodies in human vitreous samples relative to AAV2 and AAV2.7m8. Taken together, this study further contributed to our understanding of the selective pressures associated with retinal transduction via the vitreous and identified promising novel AAV capsid variants for clinical consideration.
Topics: Humans; Mice; Animals; Antibodies, Neutralizing; Capsid; Dependovirus; Intravitreal Injections; Transduction, Genetic; Primates; Capsid Proteins; Genetic Vectors
PubMed: 37814449
DOI: 10.1016/j.ymthe.2023.10.001 -
Heart, Lung & Circulation Jul 2023Globally, adeno-associated virus (AAV) vectors have been increasingly used for clinical gene therapy trials. In Australia, AAV-based gene therapy is available for... (Review)
Review
Globally, adeno-associated virus (AAV) vectors have been increasingly used for clinical gene therapy trials. In Australia, AAV-based gene therapy is available for hereditary diseases such as retinal dystrophy or spinal muscular atrophy 1 (SMA1). Many preclinical studies have used AAV vectors for gene therapy in models of cardiac disease with outcomes of varying translational potential. However, major barriers to effective and safe therapeutic gene delivery to the human heart remain to be overcome. These include tropism, efficient gene transfer, mitigating off-target gene delivery and avoidance of the host immune response. Developing such an enhanced AAV vector for cardiac gene therapy is of great interest to the field of advanced cardiac therapeutics. In this review, we provide an overview of the approaches currently being employed in the search for cardiac cell-specific AAV capsids, ranging from natural AAVs selected as a result of infection and latency in the heart, to the use of cutting-edge molecular techniques to engineer and select AAVs specific for cardiac cells with the use of high-throughput methods.
Topics: Humans; Gene Transfer Techniques; Dependovirus; Viral Tropism; Genetic Vectors
PubMed: 37451880
DOI: 10.1016/j.hlc.2023.06.704 -
BMC Cancer Dec 2023The aim of this study is to evaluate an AAV vector that can selectively target breast cancer cells and to investigate its specificity and anti-tumor effects on breast...
OBJECTIVE
The aim of this study is to evaluate an AAV vector that can selectively target breast cancer cells and to investigate its specificity and anti-tumor effects on breast cancer cells both in vitro and in vivo, offering a new therapeutic approach for the treatment of EpCAM-positive breast cancer.
METHODS
In this study, a modified AAV2 viral vector was used, in which EpCAM-specific DARPin EC1 was fused to the VP2 protein of AAV2, creating a viral vector that can target breast cancer cells. The targeting ability and anti-tumor effects of this viral vector were evaluated through in vitro and in vivo experiments.
RESULTS
The experimental results showed that the AAV2M virus could specifically infect EpCAM-positive breast cancer cells and accurately deliver the suicide gene HSV-TK to tumor tissue in mice, significantly inhibiting tumor growth. Compared to the traditional AAV2 viral vector, the AAV2M virus exhibited reduced accumulation in liver tissue and had no impact on tumor growth.
CONCLUSION
This study demonstrates that AAV2M is a gene delivery vector capable of targeting breast cancer cells and achieving selective targeting in mice. The findings offer a potential gene delivery system and strategies for gene therapy targeting EpCAM-positive breast cancer and other tumor types.
Topics: Humans; Mice; Animals; Female; Epithelial Cell Adhesion Molecule; Designed Ankyrin Repeat Proteins; Breast Neoplasms; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Dependovirus
PubMed: 38082377
DOI: 10.1186/s12885-023-11705-5 -
Clinical and Translational Medicine Mar 2024Adeno-associated virus (AAV)-based therapies are recognized as one of the most potent next-generation treatments for inherited and genetic diseases. However, several... (Review)
Review
Adeno-associated virus (AAV)-based therapies are recognized as one of the most potent next-generation treatments for inherited and genetic diseases. However, several biological and technological aspects of AAV vectors remain a critical issue for their widespread clinical application. Among them, the limited capacity of the AAV genome significantly hinders the development of AAV-based gene therapy. In this context, genetically modified transgenes compatible with AAV are opening up new opportunities for unlimited gene therapies for many genetic disorders. Recent advances in de novo protein design and remodelling are paving the way for new, more efficient and targeted gene therapeutics. Using computational and genetic tools, AAV expression cassette and transgenic DNA can be split, miniaturized, shuffled or created from scratch to mediate efficient gene transfer into targeted cells. In this review, we highlight recent advances in AAV-based gene therapy with a focus on its use in translational research. We summarize recent research and development in gene therapy, with an emphasis on large transgenes (>4.8 kb) and optimizing strategies applied by biomedical companies in the research pipeline. We critically discuss the prospects for AAV-based treatment and some emerging challenges. We anticipate that the continued development of novel computational tools will lead to rapid advances in basic gene therapy research and translational studies.
Topics: Dependovirus; Transgenes; Genetic Therapy; Genetic Vectors
PubMed: 38488469
DOI: 10.1002/ctm2.1607 -
European Journal of Pharmaceutics and... Aug 2023Development and manufacturing adeno-associated virus (AAV)-based vectors for gene therapy requires suitable analytical methods to assess the quality of the formulations...
Development and manufacturing adeno-associated virus (AAV)-based vectors for gene therapy requires suitable analytical methods to assess the quality of the formulations during development, as well as the quality of different batches and the consistency of the processes. Here, we compare biophysical methods to characterize purity and DNA content of viral capsids from five different serotypes (AAV2, AAV5, AAV6, AAV8, and AAV9). For this purpose, we apply multiwavelength sedimentation velocity analytical ultracentrifugation (SV-AUC) to obtain the species' contents and to derive the wavelength-specific correction factors for the respective insert-size. In an orthogonal manner we perform anion exchange chromatography (AEX) and UV-spectroscopy and the three methods yield comparable results on empty/filled capsid contents with these correction factors. Whereas AEX and UV-spectroscopy can quantify empty and filled AAVs, only SV-AUC could identify the low amounts of partially filled capsids present in the samples used in this study. Finally, we employ negative-staining transmission electron microscopy and mass photometry to support the empty/filled ratios with methods that classify individual capsids. The obtained ratios are consistent throughout the orthogonal approaches as long as no other impurities and aggregates are present. Our results show that the combination of selected orthogonal methods can deliver consistent empty/filled contents on non-standard genome sizes, as well as information on other relevant critical quality attributes, such as AAV capsid concentration, genome concentration, insert size length and sample purity to characterize and compare AAV preparations.
Topics: Capsid; Dependovirus; Genetic Vectors; Capsid Proteins; Ultracentrifugation; DNA
PubMed: 37196871
DOI: 10.1016/j.ejpb.2023.05.011 -
Oncotarget Nov 2023
Topics: Humans; Clustered Regularly Interspaced Short Palindromic Repeats; Gene Editing; CRISPR-Cas Systems; Dependovirus
PubMed: 38010820
DOI: 10.18632/oncotarget.28459 -
Journal of Hepatology Feb 2024Gene therapy has garnered increasing interest over recent decades. Several therapies employing gene transfer mechanisms have been developed, and, of these,... (Review)
Review
Gene therapy has garnered increasing interest over recent decades. Several therapies employing gene transfer mechanisms have been developed, and, of these, adeno-associated virus (AAV) vectors have demonstrated viability for use with in vivo gene therapy. Several AAV-based therapeutics have received regulatory approval in the last few years including those for retinal disease, spinal muscular atrophy or aromatic L-amino acid decarboxylase deficiency. Lately, with the introduction of novel liver-directed AAV vector-based therapeutics for the treatment of haemophilia A and B, gene therapy has attracted significant attention in the hepatology community, with the liver increasingly recognised as a target for gene therapy. However, the introduction of foreign DNA into hepatocytes is associated with a risk of hepatic reactions, with raised ALT (alanine aminotransferase) and AST (aspartate aminotransferase) being - so far - the most commonly reported side effects. The complete mechanisms underlying the ALT flairs remain to be determined and the long-term risks associated with these new treatments is not yet known. The liver community is increasingly being asked to support liver-directed gene therapy to mitigate potential liver associated harm. In this review, we focus on AAV vector-based gene therapy, shedding light on this promising technique and its remarkable success in haemophilia, with a special focus on hepatic complications and their management in daily clinical practice.
Topics: Humans; Gene Transfer Techniques; Dependovirus; Gastroenterologists; Genetic Therapy; Liver; Genetic Vectors
PubMed: 37890721
DOI: 10.1016/j.jhep.2023.10.029 -
Biology Open Sep 2023Robust expression of shortened, functional dystrophin provided impetus to develop adeno-associated virus (AAV)-based constructs for clinical application. Because several...
Robust expression of shortened, functional dystrophin provided impetus to develop adeno-associated virus (AAV)-based constructs for clinical application. Because several cassettes are being tested in clinical trials, this study compared the efficacies of four shortened dystrophin-promoter combinations with implications for outcomes in clinical trials: MHCK7 or MCK promoter with a shortened dystrophin transgene containing the N-terminus and spectrin repeats R1, R2, R3 and R24 (rAAVrh74.MHCK7.micro-dystrophin and rAAVrh74.MCK.micro-dystrophin, respectively); shortened dystrophin construct containing the neuronal nitric oxide (nNOS) binding site (rAAVrh74.MHCK7.DV.mini-dystrophin); and shortened dystrophin containing the C-terminus (rAAVrh74.MHCK7.micro-dystrophin.Cterm). Functional and histological benefit were examined at 4 weeks following intramuscular delivery in mdx mice. rAAVrh74.MHCK7.micro-dystrophin provided the most robust transgene expression and significantly increased specific force output in the tibialis anterior muscle. Muscle environment was normalized (i.e. reductions in central nucleation), indicating functional and histological advantages of rAAVrh74.MHCK7.micro-dystrophin. Thus, promoter choice and transgene design are critical for optimal dystrophin expression/distribution for maximal functional improvement.
Topics: Mice; Animals; Dystrophin; Muscular Dystrophy, Duchenne; Mice, Inbred mdx; Dependovirus; Actin Cytoskeleton; Disease Models, Animal
PubMed: 37670674
DOI: 10.1242/bio.059797