We have characterized the ability of adeno-associated virus (AAV) serotypes 1–9 in addition to nineteen novel vectors isolated from various tissues, to transduce mouse and human ciliated airway ...epithelium (HAE). Vectors expressing α-1-antitrypsin (AAT) and β-galactosidase were co-instilled into the mouse lung. Of all the vectors tested rh.64R1, AAV5 and AAV6 were the most efficient. The high transduction observed in mouse was reproduced in HAE cell cultures for both rh.64R1 and AAV6 but not for AAV5. Since AAV6 was the most efficient vector in mouse and HAE we also tested the transduction efficiencies of the AAV6 singleton vectors (i.e., AAV6 variants with targeted mutations) in these models. Of these, AAV6.2 transduced mouse airway epithelium and HAE with greater efficiency than all other AAV vectors tested. We demonstrated that AAV6.2 exhibits improved transduction efficiency compared to previously reported AAVs in mouse airways and in culture models of human airway epithelium and that this vector requires further development for preclinical and clinical testing.
Adeno-associated virus (AAV) vectors are promising clinical candidates for therapeutic gene transfer, and a number of AAV-based drugs may emerge on the market over the coming years. To insure the ...consistency in efficacy and safety of any drug vial that reaches the patient, regulatory agencies require extensive characterization of the final product. Identity is a key characteristic of a therapeutic product, as it ensures its proper labeling and batch-to-batch consistency. Currently, there is no facile, fast, and robust characterization assay enabling to probe the identity of AAV products at the protein level. Here, we investigated whether the thermostability of AAV particles could inform us on the composition of vector preparations. AAV-ID, an assay based on differential scanning fluorimetry (DSF), was evaluated in two AAV research laboratories for specificity, sensitivity, and reproducibility, for six different serotypes (AAV1, 2, 5, 6.2, 8, and 9), using 67 randomly selected AAV preparations. In addition to enabling discrimination of AAV serotypes based on their melting temperatures, the obtained fluorescent fingerprints also provided information on sample homogeneity, particle concentration, and buffer composition. Our data support the use of AAV-ID as a reproducible, fast, and low-cost method to ensure batch-to-batch consistency in manufacturing facilities and academic laboratories.
•Protein-based identity assay for AAV preparations•pH-induced changes in capsid thermostability•Correlation between capsid thermostability and capsid functions
Assessment of the identity of AAV-based therapeutic products is challenging. Pacouret et al. present and characterize a simple method, based on capsid thermostability, enabling to probe the identity of intact AAV particles in their final formulation.
Retinal gene therapy has come a long way in the last few decades and the development and improvement of new gene delivery technologies has been exponential. The recent promising results from the ...first clinical trials for inherited retinal degeneration due to mutations in
have provided a major breakthrough in the field and have helped cement the use of recombinant adeno-associated viruses (AAV) as the major tool for retinal gene supplementation. One of the key problems of AAV however, is its limited capacity for packaging genomic information to a maximum of around 4.8 kb. Previous studies have demonstrated that homologous recombination and/or inverted terminal repeat (ITR) mediated concatemerization of two overlapping AAV vectors can partially overcome the size limitation and help deliver larger transgenes. The aim of this study was to investigate and compare the use of different AAV dual-vector approaches in the mouse retina using a systematic approach comparing efficiencies
and
using a unique oversized reporter construct. We show that the hybrid approach relying on vector genome concatemerization by highly recombinogenic sequences and ITRs sequence overlap offers the best levels of reconstitution both
and
compared to
-splicing and overlap strategies. Our data also demonstrate that dose and vector serotype do not affect reconstitution efficiency but a discrepancy between mRNA and protein expression data suggests a bottleneck affecting translation.
The adeno-associated virus (AAV) serves as a broadly used vector system for
gene delivery. The process of AAV capsid assembly remains poorly understood. The viral cofactor assembly-activating protein ...(AAP) is required for maximum AAV production and has multiple roles in capsid assembly, namely, trafficking of the structural proteins (VP) to the nuclear site of assembly, promoting the stability of VP against multiple degradation pathways, and facilitating stable interactions between VP monomers. The N-terminal 60 amino acids of AAP (AAPN) are essential for these functions. Presumably, AAP must physically interact with VP to execute its multiple functions, but the molecular nature of the AAP-VP interaction is not well understood. Here, we query how structurally related AAVs functionally engage AAP from AAV serotype 2 (AAP2) toward virion assembly. These studies led to the identification of key residues on the lumenal capsid surface that are important for AAP-VP and for VP-VP interactions. Replacing a cluster of glutamic acid residues with a glutamine-rich motif on the conserved VP beta-barrel structure of variants incompatible with AAP2 creates a gain-of-function mutant compatible with AAP2. Conversely, mutating positively charged residues within the hydrophobic region of AAP2 and conserved core domains within AAPN creates a gain-of-function AAP2 mutant that rescues assembly of the incompatible variant. Our results suggest a model for capsid assembly where surface charge/neutrality dictates an interaction between AAPN and the lumenal VP surface to nucleate capsid assembly.
Efforts to engineer the AAV capsid to gain desirable properties for gene therapy (e.g., tropism, reduced immunogenicity, and higher potency) require that capsid modifications do not affect particle assembly. The relationship between VP and the cofactor that facilitates its assembly, AAP, is central to both assembly preservation and vector production. Understanding the requirements for this compatibility can inform manufacturing strategies to maximize production and reduce costs. Additionally, library-based approaches that simultaneously examine a large number of capsid variants would benefit from a universally functional AAP, which could hedge against overlooking variants with potentially valuable phenotypes that were lost during vector library production due to incompatibility with the cognate AAP. Studying interactions between the structural and nonstructural components of AAV enhances our fundamental knowledge of capsid assembly mechanisms and the protein-protein interactions required for productive assembly of the icosahedral capsid.
Leber congenital amaurosis (LCA) is a severe disorder resulting in visual impairment usually starting in the first year of life. The most frequent genetic cause of LCA is an intronic mutation in ...CEP290 (c.2991 + 1655A > G) that creates a cryptic splice donor site resulting in the insertion of a pseudoexon (exon X) into CEP290 mRNA. Previously, we showed that naked antisense oligonucleotides (AONs) effectively restored normal CEP290 splicing in patient-derived lymphoblastoid cells. We here explore the therapeutic potential of naked and adeno-associated virus (AAV)-packaged AONs in vitro and in vivo In both cases, AON delivery fully restored CEP290 pre-mRNA splicing, significantly increased CEP290 protein levels and rescued a ciliary phenotype present in patient-derived fibroblast cells. Moreover, administration of naked and AAV-packaged AONs to the retina of a humanized mutant Cep290 mouse model, carrying the intronic mutation, showed a statistically significant reduction of exon X-containing Cep290 transcripts, without compromising the retinal structure. Together, our data highlight the tremendous therapeutic prospective of AONs for the treatment of not only CEP290-associated LCA but potentially many other subtypes of retinal dystrophy caused by splicing mutations.
Gene therapy is emerging as a therapeutic modality for treating disorders of the retina. Photoreceptor cells are the primary cell type affected in many inherited diseases of retinal degeneration. ...Successfully treating these diseases with gene therapy requires the identification of efficient and safe targeting vectors that can transduce photoreceptor cells. One serotype of adeno-associated virus, AAV2, has been used successfully in clinical trials to treat a form of congenital blindness that requires transduction of the supporting cells of the retina in the retinal pigment epithelium (RPE). Here, we determined the dose required to achieve targeting of AAV2 and AAV8 vectors to photoreceptors in nonhuman primates. Transgene expression in animals injected subretinally with various doses of AAV2 or AAV8 vectors carrying a green fluorescent protein transgene was correlated with surgical, clinical, and immunological observations. Both AAV2 and AAV8 demonstrated efficient transduction of RPE, but AAV8 was markedly better at targeting photoreceptor cells. These preclinical results provide guidance for optimal vector and dose selection in future human gene therapy trials to treat retinal diseases caused by loss of photoreceptors.
The field of gene therapy for retinal blinding disorders is experiencing incredible momentum, justified by hopeful results in early stage clinical trials for inherited retinal degenerations. The ...premise of the use of the gene as a drug has come a long way, and may have found its niche in the treatment of retinal disease. Indeed, with only limited treatment options available for retinal indications, gene therapy has been proven feasible, safe, and effective and may lead to durable effects following a single injection. Here, we aim at putting into context the promise and potential, the technical, clinical, and economic boundaries limiting its application and development, and speculate on a future in which gene therapy is an integral component of ophthalmic clinical care.
New recombinant serotypes of AAV vectors Gao, Guangping; Vandenberghe, Luk H; Wilson, James M
Current gene therapy,
06/2005, Volume:
5, Issue:
3
Journal Article
Peer reviewed
AAV based vectors can achieve stable gene transfer with minimal vector related toxicities. AAV serotype 2 (AAV2) is the first AAV that was vectored for gene transfer applications. However, the ...restricted tissue tropism of AAV and its low transduction efficiency have limited its further development as vector. Recent studies using vectors derived from alternative AAV serotypes such as AAV1, 4, 5 and 6 have shown improved potency and broadened tropism of the AAV vector by packaging the same vector genome with different AAV capsids. In an attempt to search for potent AAV vectors with enhanced performance profiles, molecular techniques were employed for the detection and isolation of endogenous AAVs from a variety of human and non-human primate (NHP) tissues. A family of novel primate AAVs consisting of 110 non-redundant species of proviral sequences was discovered and turned to be prevalent in 18-19% of the tissues evaluated. Phylogenetic and functional analyses revealed that primate AAVs are segregated into clades based on phylogenetic relatedness. The members within a clade share functional and serological properties. Initial evaluation in mouse models of vectors based on these novel AAVs for tissue tropism and gene transfer potency led to the identification of some vector with improved gene transfer to different target tissues. Gene therapy treatment of several mouse and canine models with novel AAV vectors achieved long term phenotypic corrections. Vectors based on new primate AAVs could become the next generation of efficient gene transfer vehicles for various gene therapy applications.
Not only is the vector formulation deposited below the retina by iatrogenically producing a retinotomy and self-resolving retinal detachment, but it is preceded by the partial removal of the vitreous ...jelly to facilitate the retinal maneuver. Since these interventions have the potential to harm the frail XLRS retina, an intravitreal injection would be highly preferred, as Cukras and colleagues3 pursued here. Intravitreal injection has the potential advantage of reaching a broad area of retina, whereas the effect of a subretinal injection is largely retained within the bleb, i.e., the subretinal space generated by the injection. ...intravitreal injection of gene therapy agents is theoretically highly preferable over a subretinal approach, not just for XLRS, but arguably for most retinal gene therapy disease targets given its limited invasiveness, its ability for widespread retinal targeting, and the clinical experience and comfort practitioners have garnered with the procedure over the past decades. ...gene transfer from the vitreous space into the retina in large eyes is clearly inefficient, in need of high dosing, and broader therapeutic success likely will hinge on development of more potent and better tolerated vector systems.Conflicts of Interest J.W.M. is a consultant and advisor for Genentech/Roche, Bausch + Lomb, Kalvista Pharmaceuticals, and ONL Therapeutics; has equity in ONL Therapeutics; and has patents and/or royalties from ONL Therapeutics and Massachussettes Eye and Ear and Valeant Pharmaceuticals and Massachusettes Eye and Ear.
Diseases of the nervous system have devastating effects and are widely distributed among the population, being especially prevalent in the elderly. These diseases are often caused by inherited ...genetic mutations that result in abnormal nervous system development, neurodegeneration, or impaired neuronal function. Other causes of neurological diseases include genetic and epigenetic changes induced by environmental insults, injury, disease-related events or inflammatory processes. Standard medical and surgical practice has not proved effective in curing or treating these diseases, and appropriate pharmaceuticals do not exist or are insufficient to slow disease progression. Gene therapy is emerging as a powerful approach with potential to treat and even cure some of the most common diseases of the nervous system. Gene therapy for neurological diseases has been made possible through progress in understanding the underlying disease mechanisms, particularly those involving sensory neurons, and also by improvement of gene vector design, therapeutic gene selection, and methods of delivery. Progress in the field has renewed our optimism for gene therapy as a treatment modality that can be used by neurologists, ophthalmologists and neurosurgeons. In this Review, we describe the promising gene therapy strategies that have the potential to treat patients with neurological diseases and discuss prospects for future development of gene therapy.
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