RESUMEN

Intervertebral disc (IVD) degeneration damaging the extracellular matrix (ECM) of IVDs is the main cause of spine-associated disorders. Degenerative disc disease (DDD) is a multifaceted disorder, where environmental factors, inflammatory cytokines and catabolic enzymes act together. DDD starts typically due to imbalance between ECM biosynthesis and degradation within IVDs, especially through unbalanced degradation of aggrecan and collagen II in nucleus pulposus (NP). Current treatment approaches are primarily based on conservative or surgical therapies, which are insufficient for biological regeneration. The disintegrins and metalloproteinases with thrombospondin motifs (ADAMTSs) and matrix metalloproteinases (MMPs) are the key proteolytic enzymes for degradation of aggrecan and collagens. Previously, high expression levels of ADAMTS4, ADAMTS5, MMP3 and MMP13, which are accompanied with low levels of aggrecan and collagen II, were demonstrated in degenerative human NP cells. Moreover, self-complementary adeno-associated virus type 6 (scAAV6) mediated inhibitions of ADAMTS4 and ADAMTS5 by RNA-interference (RNAi) could specifically enhance aggrecan level. Thus, MMPs are apparently the main degrading enzymes of collagen II in NP. Furthermore, scAAV6-mediated inhibitions of MMP3 and MMP13 have not yet been investigated. Therefore, we attempted to enhance the level of collagen II in degenerative NP cells by scAAV6-RNAi-mediated inhibitions of MMP3 and MMP13. MRI was used to determine preoperative grading of IVD degeneration in patients. After isolation and culturing of NP cells, cells were transduced with scAAV6-shRNAs targeting MMP3 or MMP13; and analysed by fluorescence microscopy, FACS, MTT assay, RT-qPCR, ELISA and western blotting. scAAV6-shRNRs have no impact on cell viability and proliferation, despite high transduction efficiencies (98.6%) and transduction units (1383 TU/Cell). Combined knockdown of MMP3 (92.8%) and MMP13 (90.9%) resulted in highest enhancement of collagen II (143.2%), whereby treatment effects were significant over 56 days (p < 0.001). Conclusively, scAAV6-RNAi-mediated inhibitions of MMP3 and MMP13 help to progress less immunogenic and enduring biological treatments in DDD.

Asunto(s)

Proteína ADAMTS4 , Degeneración del Disco Intervertebral , Metaloproteinasa 13 de la Matriz , Metaloproteinasa 3 de la Matriz , Núcleo Pulposo , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/genética , Humanos , Metaloproteinasa 13 de la Matriz/metabolismo , Degeneración del Disco Intervertebral/metabolismo , Degeneración del Disco Intervertebral/terapia , Degeneración del Disco Intervertebral/patología , Degeneración del Disco Intervertebral/genética , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patología , Proteína ADAMTS4/metabolismo , Proteína ADAMTS4/genética , Colágeno Tipo II/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Proteína ADAMTS5/metabolismo , Proteína ADAMTS5/genética , Interferencia de ARN , Células Cultivadas , Agrecanos/metabolismo

RESUMEN

Aging is a complex multifactorial process associated with epigenome dysregulation, increased cellular senescence, and decreased rejuvenation capacity. Short-term cyclic expression of octamer-binding transcription factor 4 (Oct4), sex-determining region Y-box 2 (Sox2), Kruppel-like factor 4 (Klf4), and cellular myelocytomatosis oncogene (cMyc) (OSKM) in wild-type mice improves health but fails to distinguish cell states, posing risks to healthy cells. Here, we delivered a single dose of adeno-associated viruses (AAVs) harboring OSK under the control of the cyclin-dependent kinase inhibitor 2a (Cdkn2a) promoter to specifically partially reprogram aged and stressed cells in a mouse model of Hutchinson-Gilford progeria syndrome (HGPS). Mice showed reduced expression of proinflammatory cytokines and extended life spans upon aged cell-specific OSK expression. The bone marrow and spleen, in particular, showed pronounced gene expression changes, and partial reprogramming in aged HGPS mice led to a shift in the cellular composition of the hematopoietic stem cell compartment toward that of young mice. Administration of AAVs carrying Cdkn2a-OSK to naturally aged wild-type mice also delayed aging phenotypes and extended life spans without altering the incidence of tumor development. Furthermore, intradermal injection of AAVs carrying Cdkn2a-OSK led to improved wound healing in aged wild-type mice. Expression of CDKN2A-OSK in aging or stressed human primary fibroblasts led to reduced expression of inflammation-related genes but did not alter the expression of cell cycle-related genes. This targeted partial reprogramming approach may therefore facilitate the development of strategies to improve health and life span and enhance resilience in the elderly.

Asunto(s)

Envejecimiento , Reprogramación Celular , Senescencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Modelos Animales de Enfermedad , Factor 4 Similar a Kruppel , Animales , Factor 4 Similar a Kruppel/metabolismo , Envejecimiento/metabolismo , Ratones , Humanos , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Biomarcadores/metabolismo , Progeria/metabolismo , Progeria/genética , Progeria/patología , Dependovirus/metabolismo , Regiones Promotoras Genéticas/genética

RESUMEN

Adeno-associated viruses (AAVs) are foundational gene delivery tools for basic science and clinical therapeutics. However, lack of mechanistic insight, especially for engineered vectors created by directed evolution, can hamper their application. Here, we adapt an unbiased human cell microarray platform to determine the extracellular and cell surface interactomes of natural and engineered AAVs. We identify a naturally-evolved and serotype-specific interaction between the AAV9 capsid and human interleukin 3 (IL3), with possible roles in host immune modulation, as well as lab-evolved low-density lipoprotein receptor-related protein 6 (LRP6) interactions specific to engineered capsids with enhanced blood-brain barrier crossing in non-human primates after intravenous administration. The unbiased cell microarray screening approach also allows us to identify off-target tissue binding interactions of engineered brain-enriched AAV capsids that may inform vectors' peripheral organ tropism and side effects. Our cryo-electron tomography and AlphaFold modeling of capsid-interactor complexes reveal LRP6 and IL3 binding sites. These results allow confident application of engineered AAVs in diverse organisms and unlock future target-informed engineering of improved viral and non-viral vectors for non-invasive therapeutic delivery to the brain.

Asunto(s)

Barrera Hematoencefálica , Dependovirus , Interleucina-3 , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , Transcitosis , Animales , Humanos , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Cápside/metabolismo , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Dependovirus/genética , Dependovirus/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/administración & dosificación , Células HEK293 , Interleucina-3/metabolismo , Unión Proteica , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo

RESUMEN

OBJECTIVE: Recombinant adeno-associated virus (rAAV) vectors are powerful tools for the sustained expression of proteins in vivo and have been successfully used for mechanistic studies in mice. A major challenge associated with this method is to obtain tissue specificity and high expression levels without need of local virus administration. METHODS: To achieve this goal for brown adipose tissue (BAT), we developed a rAAV vector for intravenous bolus injection, which includes an expression cassette comprising an uncoupling protein-1 enhancer-promoter for transcription in brown adipocytes and miR122 target sequences for suppression of expression in the liver, combined with packaging in serotype Rec2 capsid protein. To test tissue specificity, we used a version of this vector expressing Cre recombinase to transduce mice with floxed alleles to knock out MLXIPL (ChREBP) or tdTomato-Cre reporter mice. RESULTS: We demonstrated efficient Cre-dependent recombination in interscapular BAT and variable effects in minor BAT depots, but little or no efficacy in white adipose tissues, liver and other organs. Direct overexpression of glucose transporter SLC2A1 (GLUT1) using the rAAV vector in wild type mice resulted in increased glucose uptake and glucose-dependent gene expression in BAT, indicating usefulness of this vector to increase the function even of abundant proteins. CONCLUSION: Taken together, we describe a novel brown adipocyte-specific rAAV method to express proteins for loss-of-function and gain-of-function metabolic studies. The approach will enable researchers to access brown fat swiftly, reduce animal breeding time and costs, as well as enable the creation of new transgenic mouse models combining multiple transgenes.

Asunto(s)

Adipocitos Marrones , Tejido Adiposo Pardo , Dependovirus , Vectores Genéticos , Animales , Ratones , Dependovirus/genética , Dependovirus/metabolismo , Vectores Genéticos/genética , Adipocitos Marrones/metabolismo , Tejido Adiposo Pardo/metabolismo , Ratones Endogámicos C57BL , Inyecciones Intravenosas , Masculino , Serogrupo , Proteína Desacopladora 1/metabolismo , Proteína Desacopladora 1/genética , Integrasas/metabolismo , Integrasas/genética

RESUMEN

Gene replacement using adeno-associated virus (AAV) vectors is a promising therapeutic approach for many diseases1,2. However, this therapeutic modality is challenged by the packaging capacity of AAVs (approximately 4.7 kilobases)3, limiting its application for disorders involving large coding sequences, such as Duchenne muscular dystrophy, with a 14 kilobase messenger RNA. Here we developed a new method for expressing large dystrophins by utilizing the protein trans-splicing mechanism mediated by split inteins. We identified several split intein pairs that efficiently join two or three fragments to generate a large midi-dystrophin or the full-length protein. We show that delivery of two or three AAVs into dystrophic mice results in robust expression of large dystrophins and significant physiological improvements compared with micro-dystrophins. Moreover, using the potent myotropic AAVMYO4, we demonstrate that low total doses (2 × 1013 viral genomes per kg) are sufficient to express large dystrophins in striated muscles body-wide with significant physiological corrections in dystrophic mice. Our data show a clear functional superiority of large dystrophins over micro-dystrophins that are being tested in clinical trials. This method could benefit many patients with Duchenne or Becker muscular dystrophy, regardless of genotype, and could be adapted to numerous other disorders caused by mutations in large genes that exceed the AAV capacity.

Asunto(s)

Distrofina , Terapia Genética , Inteínas , Distrofia Muscular de Duchenne , Empalme de Proteína , Animales , Humanos , Masculino , Ratones , Dependovirus/genética , Dependovirus/metabolismo , Modelos Animales de Enfermedad , Distrofina/biosíntesis , Distrofina/deficiencia , Distrofina/genética , Distrofina/metabolismo , Terapia Genética/métodos , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Inteínas/genética , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Distrofia Muscular de Duchenne/metabolismo , Empalme de Proteína/genética

RESUMEN

Progranulin (PGRN) haploinsufficiency is a major risk factor for frontotemporal lobar degeneration with TAR DNA-binding protein 43 (TDP-43) pathology (FTLD-GRN). Multiple therapeutic strategies are in clinical development to restore PGRN in the CNS, including gene therapy. However, a limitation of current gene therapy approaches aimed to alleviate FTLD-associated pathologies may be their inefficient brain exposure and biodistribution. We therefore developed an adeno-associated virus (AAV) targeting the liver (L) to achieve sustained peripheral expression of a transferrin receptor (TfR) binding, brain-penetrant (b) PGRN variant [AAV(L):bPGRN] in two mouse models of FTLD-GRN, namely, Grn knockout and GrnxTmem106b double knockout mice. This therapeutic strategy avoids potential safety and biodistribution issues of CNS-administered AAVs and maintains sustained concentrations of PGRN in the brain after a single dose. AAV(L):bPGRN treatment reduced several FTLD-GRN-associated pathologies including severe motor function deficits, aberrant TDP-43 phosphorylation, dysfunctional protein degradation, lipid metabolism, gliosis, and neurodegeneration in the brain. The potential translatability of our findings was tested in an in vitro model using cocultured human induced pluripotent stem cell (hiPSC)-derived microglia lacking PGRN and TMEM106B and wild-type hiPSC-derived neurons. As in mice, aberrant TDP-43, lysosomal dysfunction, and neuronal loss were ameliorated after treatment with exogenous TfR-binding protein transport vehicle fused to PGRN (PTV:PGRN). Together, our studies suggest that peripherally administered brain-penetrant PGRN replacement strategies ameliorate FTLD-GRN relevant phenotypes including TDP-43 pathology, neurodegeneration, and behavioral deficits. Our data provide preclinical proof of concept for the use of this AAV platform for treatment of FTLD-GRN and potentially other CNS disorders.

Asunto(s)

Encéfalo , Dependovirus , Modelos Animales de Enfermedad , Degeneración Lobar Frontotemporal , Ratones Noqueados , Progranulinas , Animales , Humanos , Ratones , Encéfalo/metabolismo , Encéfalo/patología , Dependovirus/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Degeneración Lobar Frontotemporal/metabolismo , Degeneración Lobar Frontotemporal/patología , Terapia Genética , Fosforilación , Progranulinas/metabolismo , Progranulinas/genética , Receptores de Transferrina/metabolismo

RESUMEN

Single-cell-type proteomics is an emerging field of research that combines cell-type specificity with the comprehensive proteome coverage offered by bulk proteomics. However, the extraction of single-cell-type proteomes remains a challenge, particularly for hard-to-isolate cells like neurons. In this chapter, we present an innovative technique for profiling single-cell-type proteomes using adeno-associated virus (AAV)-mediated proximity labeling (PL) and tandem-mass-tag (TMT) mass spectrometry. This technique eliminates the need for cell isolation and offers a streamlined workflow, including AAV delivery to express TurboID (an engineered biotin ligase) controlled by cell-type-specific promoters, biotinylated protein purification, on-bead digestion, TMT labeling, and liquid chromatography-mass spectrometry (LC-MS). We examined this method by analyzing distinct brain cell types in mice. Initially, recombinant AAVs were used to concurrently express TurboID and mCherry proteins driven by neuron- or astrocyte-specific promoters, which was validated through co-immunostaining with cellular markers. With biotin purification and TMT analysis, we successfully identified around 10,000 unique proteins from a few micrograms of protein samples with high reproducibility. Our statistical analyses revealed that these proteomes encompass cell-type-specific cellular pathways. By utilizing this technique, researchers can explore the proteomic landscape of specific cell types, paving the way for new insights into cellular processes, deciphering disease mechanisms, and identifying therapeutic targets in neuroscience and beyond.

Asunto(s)

Encéfalo , Dependovirus , Proteoma , Proteómica , Espectrometría de Masas en Tándem , Dependovirus/genética , Dependovirus/metabolismo , Animales , Ratones , Proteómica/métodos , Proteoma/análisis , Encéfalo/metabolismo , Espectrometría de Masas en Tándem/métodos , Análisis de la Célula Individual/métodos , Neuronas/metabolismo , Cromatografía Liquida/métodos , Vectores Genéticos/genética , Biotinilación , Espectrometría de Masas/métodos , Astrocitos/metabolismo

RESUMEN

Adeno-associated viruses (AAVs) package a single-stranded (ss) DNA genome of 4.7 kb in their capsid of ~20 nm in diameter. AAV replication requires co-infection of a helper virus, such as adenovirus. During the optimization of recombinant AAV production, a small viral nonstructural protein, membrane-associated accessory protein (MAAP), was identified. However, the function of the MAAP in the context of AAV infection remains unknown. Here, we investigated the expression strategy and function of the MAAP during infection of both AAV2 and AAV5 in human embryonic kidney (HEK)293 cells. We found that AAV2 MAAP2 and AAV5 MAAP5 are expressed from the capsid gene (cap)-transcribing mRNA spliced from the donor to the second splice site that encodes VP2 and VP3. Thus, this AAV cap gene transcribes a multicistronic mRNA that can be translated to four viral proteins, MAAP, VP2, AAP, and VP3 in order. In AAV2 infection, MAAP2 predominantly localized in the cytoplasm, alongside the capsid, near the nuclear and plasma membranes, but a fraction of MAAP2 exhibited nuclear localization. In AAV5 infection, MAAP5 revealed a distinct pattern, predominantly localizing within the nucleus. In the cells infected with an MAAP knockout mutant of AAV2 or AAV5, both viral DNA replication and virus replication increased, whereas virus egress decreased, and the decrease in virus egress can be restored by providing MAAP in trans. In summary, MAAP, a novel AAV nonstructural protein translated from a multicistronic viral cap mRNA, not only facilitates cellular egress of AAV but also likely negatively affects viral DNA replication during infection. IMPORTANCE: Recombinant adeno-associated virus (rAAV) has been used as a gene delivery vector in clinical gene therapy. In current gene therapies employing rAAV, a high dose of the vector is required. Consequently, there is a high demand for efficient and high-purity vector production systems. In this study, we demonstrated that membrane-associated accessory protein (MAAP), a small viral nonstructural protein, is translated from the same viral mRNA transcript encoding VP2 and VP3. In AAV-infected cells, apart from its prevalent expression in the cytoplasm with localization near the plasma and nuclear membranes, the MAAP also exhibits notable localization within the nucleus. During AAV infection, MAAP expression increases the cellular egress of progeny virions and decreases viral DNA replication and progeny virion production. Thus, the choice of MAAP expression has pros and cons during AAV infection, which could provide a guide to rAAV production.

Asunto(s)

Dependovirus , Infecciones por Parvoviridae , Proteínas no Estructurales Virales , Humanos , Proteínas de la Cápside/genética , Dependovirus/genética , Dependovirus/metabolismo , Dependovirus/fisiología , Células HEK293 , Infecciones por Parvoviridae/virología , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Replicación Viral , Genes Virales/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo

RESUMEN

A deficiency in the shortest dystrophin-gene product, Dp71, is a pivotal aggravating factor for intellectual disabilities in Duchenne muscular dystrophy (DMD). Recent advances in preclinical research have achieved some success in compensating both muscle and brain dysfunctions associated with DMD, notably using exon skipping strategies. However, this has not been studied for distal mutations in the DMD gene leading to Dp71 loss. In this study, we aimed to restore brain Dp71 expression in the Dp71-null transgenic mouse using an adeno-associated virus (AAV) administrated either by intracardiac injections at P4 (ICP4) or by bilateral intracerebroventricular (ICV) injections in adults. ICP4 delivery of the AAV9-Dp71 vector enabled the expression of 2 to 14% of brain Dp71, while ICV delivery enabled the overexpression of Dp71 in the hippocampus and cortex of adult mice, with anecdotal expression in the cerebellum. The restoration of Dp71 was mostly located in the glial endfeet that surround capillaries, and it was associated with partial localization of Dp71-associated proteins, α1-syntrophin and AQP4 water channels, suggesting proper restoration of a scaffold of proteins involved in blood-brain barrier function and water homeostasis. However, this did not result in significant improvements in behavioral disturbances displayed by Dp71-null mice. The potential and limitations of this AAV-mediated strategy are discussed. This proof-of-concept study identifies key molecular markers to estimate the efficiencies of Dp71 rescue strategies and opens new avenues for enhancing gene therapy targeting cognitive disorders associated with a subgroup of severely affected DMD patients.

Asunto(s)

Encéfalo , Dependovirus , Distrofina , Proteínas de la Membrana , Proteínas Musculares , Animales , Masculino , Ratones , Acuaporina 4/metabolismo , Acuaporina 4/genética , Conducta Animal , Encéfalo/metabolismo , Encéfalo/patología , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Dependovirus/genética , Dependovirus/metabolismo , Modelos Animales de Enfermedad , Distrofina/metabolismo , Distrofina/genética , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Ratones Endogámicos C57BL , Ratones Noqueados , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/terapia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patología

RESUMEN

Adeno-associated virus (AAV)-based gene therapies are in clinical development for haemophilia and other genetic diseases. Since the recombinant AAV genome primarily remains episomal, it provides the opportunity for long-term expression in tissues that are not proliferating and reduces the safety concerns compared with integrating viral vectors. However, AAV integration events are detected at a low frequency. Preclinical studies in mouse models have reported hepatocellular carcinoma (HCC) after systemic AAV administration in some settings, though this has not been reported in large animal models. The risk of HCC or other cancers after AAV gene therapy in clinical studies thus remains theoretical. Potential risk factors for HCC after gene therapy are beginning to be elucidated through animal studies, but their relevance to human studies remains unknown. Studies to investigate the factors that may influence the risk of oncogenesis as well as detailed investigation of cases of cancer in AAV gene therapy patients will be important to define the potential risk of AAV genotoxicity.

Asunto(s)

Carcinoma Hepatocelular , Neoplasias Hepáticas , Ratones , Animales , Humanos , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/genética , Carcinoma Hepatocelular/patología , Vectores Genéticos , Plásmidos , Terapia Genética , Dependovirus/genética , Dependovirus/metabolismo , Integración Viral

RESUMEN

Today, adeno-associated virus (AAV)-based vectors are arguably the most promising in vivo gene delivery vehicles for durable therapeutic gene expression. Advances in molecular engineering, high-throughput screening platforms, and computational techniques have resulted in a toolbox of capsid variants with enhanced performance over parental serotypes. Despite their considerable promise and emerging clinical success, there are still obstacles hindering their broader use, including limited transduction capabilities, tissue/cell type-specific tropism and penetration into tissues through anatomical barriers, off-target tissue biodistribution, intracellular degradation, immune recognition, and a lack of translatability from preclinical models to clinical settings. Here, we first describe the transduction mechanisms of natural AAV serotypes and explore the current understanding of the systemic and cellular hurdles to efficient transduction. We then outline progress in developing designer AAV capsid variants, highlighting the seminal discoveries of variants which can transduce the central nervous system upon systemic administration, and, to a lesser extent, discuss the targeting of the peripheral nervous system, eye, ear, lung, liver, heart, and skeletal muscle, emphasizing their tissue and cell specificity and translational promise. In particular, we dive deeper into the molecular mechanisms behind their enhanced properties, with a focus on their engagement with host cell receptors previously inaccessible to natural AAV serotypes. Finally, we summarize the main findings of our review and discuss future directions.

Asunto(s)

Cápside , Dependovirus , Cápside/metabolismo , Dependovirus/metabolismo , Serogrupo , Distribución Tisular , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Tropismo , Vectores Genéticos/genética

RESUMEN

Adeno-associated virus (AAV) is a member of the genus Dependoparvovirus, which infects a wide range of vertebrate species. Here, we observe that, unlike most primate AAV isolates, avian AAV is transcriptionally silenced in human cells. By swapping the VP1 N terminus from primate AAVs (e.g., AAV8) onto non-mammalian isolates (e.g., avian AAV), we identify a minimal component of the AAV capsid that controls viral transcription and unlocks robust transduction in both human cells and mouse tissue. This effect is accompanied by increased AAV genome chromatin accessibility and altered histone methylation. Proximity ligation analysis reveals that host factors are selectively recruited by the VP1 N terminus of AAV8 but not avian AAV. Notably, these include AAV essential factors implicated in the nuclear factor κB pathway, chromatin condensation, and histone methylation. We postulate that the AAV capsid has evolved mechanisms to recruit host factors to its genome, allowing transcriptional activation in a species-specific manner.

Asunto(s)

Cápside , Dependovirus , Humanos , Animales , Ratones , Cápside/metabolismo , Dependovirus/metabolismo , Histonas/metabolismo , Transcripción Viral , Vectores Genéticos , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Primates , Especificidad del Huésped , Cromatina/metabolismo

RESUMEN

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.

Asunto(s)

Dependovirus , Terapia Genética , Dependovirus/genética , Dependovirus/metabolismo , Transgenes/genética , Vectores Genéticos/genética

RESUMEN

Adeno-associated virus (AAV) requires co-infection with helper virus for efficient replication. We previously reported that Human Bocavirus 1 (HBoV1) genes, including NP1, NS2, and BocaSR, were critical for AAV2 replication. Here, we first demonstrate the essential roles of the NP1 protein in AAV2 DNA replication and protein expression. We show that NP1 binds to single-strand DNA (ssDNA) at least 30 nucleotides (nt) in length in a sequence-independent manner. Furthermore, NP1 colocalized with the BrdU-labeled AAV2 DNA replication center, and the loss of the ssDNA-binding ability of NP1 by site-directed mutation completely abolished AAV2 DNA replication. We used affinity-tagged NP1 protein to identify host cellular proteins associated with NP1 in cells cotransfected with the HBoV1 helper genes and AAV2 duplex genome. Of the identified proteins, we demonstrate that NP1 directly binds to the DBD-F domain of the RPA70 subunit with a high affinity through the residues 101-121. By reconstituting the heterotrimer protein RPA in vitro using gel filtration, we demonstrate that NP1 physically associates with RPA to form a heterologous complex characterized by typical fast-on/fast-off kinetics. Following a dominant-negative strategy, we found that NP1-RPA complex mainly plays a role in expressing AAV2 capsid protein by enhancing the transcriptional activity of the p40 promoter. Our study revealed a novel mechanism by which HBoV1 NP1 protein supports AAV2 DNA replication and capsid protein expression through its ssDNA-binding ability and direct interaction with RPA, respectively.IMPORTANCERecombinant adeno-associated virus (rAAV) vectors have been extensively used in clinical gene therapy strategies. However, a limitation of these gene therapy strategies is the efficient production of the required vectors, as AAV alone is replication-deficient in the host cells. HBoV1 provides the simplest AAV2 helper genes consisting of NP1, NS2, and BocaSR. An important question regarding the helper function of HBoV1 is whether it provides any direct function that supports AAV2 DNA replication and protein expression. Also of interest is how HBoV1 interplays with potential host factors to constitute a permissive environment for AAV2 replication. Our studies revealed that the multifunctional protein NP1 plays important roles in AAV2 DNA replication via its sequence-independent ssDNA-binding ability and in regulating AAV2 capsid protein expression by physically interacting with host protein RPA. Our findings present theoretical guidance for the future application of the HBoV1 helper genes in the rAAV vector production.

Asunto(s)

Proteínas de la Cápside , Cápside , ADN de Cadena Simple , ADN Viral , Proteínas de Unión al ADN , Dependovirus , Bocavirus Humano , Proteínas Virales , Humanos , Cápside/metabolismo , Proteínas de la Cápside/biosíntesis , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Dependovirus/genética , Dependovirus/crecimiento & desarrollo , Dependovirus/metabolismo , ADN de Cadena Simple/biosíntesis , ADN de Cadena Simple/metabolismo , ADN Viral/biosíntesis , ADN Viral/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Viral de la Expresión Génica , Bocavirus Humano/genética , Bocavirus Humano/metabolismo , Cinética , Mutagénesis Sitio-Dirigida , Mutación , Regiones Promotoras Genéticas , Unión Proteica , Dominios Proteicos , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral

RESUMEN

Adeno-associated virus (AAV) analytical characterization is crucial to the well-defined and reproducible production of human gene therapies utilizing the AAV vector modality. The establishment of analytical methods based upon technology platforms currently widely used by bio-therapeutic manufacturers, namely HPLC, will assist efforts to produce high quality AAV reproducibly and decrease chemical manufacturing and control challenges in method portability and reliability. AAV analysis by size exclusion chromatography (SEC) is currently practiced with columns and mobile phase conditions traditional to SEC of proteins. Here, an improved method to measure multiple AVV critical quality attributes (CQA) rapidly by SEC is explored. The use of short columns made with small particles at high flow rates resulted in up to 80 % reduction in analysis time and 66 % in sample consumption while maintaining reliable quantitation of AAV aggregate or high molecular weight (HMW) content. These results were demonstrated across four different AAV serotypes. Furthermore, critical AAV sample handling learnings are shared.

Asunto(s)

Dependovirus , Proteínas , Humanos , Dependovirus/genética , Dependovirus/metabolismo , Reproducibilidad de los Resultados , Proteínas/metabolismo , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Vectores Genéticos

RESUMEN

Variants within the Retinitis Pigmentosa GTPase regulator (RPGR) gene are the predominant cause of X-Linked Retinitis Pigmentosa (XLRP), a common and severe form of inherited retinal disease. XLRP is characterised by the progressive degeneration and loss of photoreceptors, leading to visual loss and, ultimately, bilateral blindness. Unfortunately, there are no effective approved treatments for RPGR-associated XLRP. We sought to investigate the efficacy of RPGRORF15 gene supplementation using a clinically relevant construct in human RPGR-deficient retinal organoids (ROs). Isogenic RPGR knockout (KO)-induced pluripotent stem cells (IPSCs) were generated using established CRISPR/Cas9 gene editing methods targeting RPGR. RPGR-KO and isogenic wild-type IPSCs were differentiated into ROs and utilised to test the adeno associated virus (AAV) RPGR (AAV-RPGR) clinical vector construct. The transduction of RPGR-KO ROs using AAV-RPGR successfully restored RPGR mRNA and protein expression and localisation to the photoreceptor connecting cilium in rod and cone photoreceptors. Vector-derived RPGR demonstrated equivalent levels of glutamylation to WT ROs. In addition, treatment with AAV-RPGR restored rhodopsin localisation within RPGR-KO ROs, reducing mislocalisation to the photoreceptor outer nuclear layer. These data provide mechanistic insights into RPGRORF15 gene supplementation functional potency in human photoreceptor cells and support the previously reported Phase I/II trial positive results using this vector construct in patients with RPGR-associated XLRP, which is currently being tested in a Phase III clinical trial.

Asunto(s)

Opsinas , Retinitis Pigmentosa , Humanos , Opsinas/genética , Dependovirus/genética , Dependovirus/metabolismo , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Portadoras/metabolismo , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/terapia , Retinitis Pigmentosa/metabolismo , Rodopsina/genética , Terapia Genética/métodos , Mutación

RESUMEN

Adeno-associated virus (AAV) is one of the most frequently used viral vectors in the field of gene therapy. However, the industrial production of AAV is facing key bottlenecks such as low yield and high-cost. The aim of this study was to establish a technology system for production of AAV in the double virus infected insects by using multiple-gene deleted baculovirus. First, a multiple gene deleted baculovirus for AAV production was constructed, and the baculovirus titer and its effect on infected cells was examined. Subsequently, the insect cells were co-infected with the double baculovirus and the infection conditions were optimized. At the final stage, we performed AAV production based on optimized conditions, and evaluated relevant parameters including production titer and quality. The results showed that the titer of AAV produced in the multiple gene deleted baculovirus was not different from that of the wild type, but the rate of cell death was significantly slower upon infection. Using the double virus route for optimized production of AAV, the genome titers were 1.63×1011 VG/mL for Bac4.0-1 and 1.02×1011 VG/mL for Bac5.0-2, which were elevated 240% and 110%, respectively, compared with that of the wild-type. Electron microscopy observations revealed that all three groups exhibited normal AAV viral morphology and they showed similar transduction activity. Taken together, we developed an AAV production system based on the infection of insect cells using multiple-gene deleted baculovirus, which significantly improved the virus yield and showed application potential.

Asunto(s)

Baculoviridae , Dependovirus , Animales , Dependovirus/genética , Dependovirus/metabolismo , Baculoviridae/genética , Baculoviridae/metabolismo , Línea Celular , Vectores Genéticos , Insectos/genética

RESUMEN

Early gene therapy clinical trials for the treatment of Haemophilia B have been instrumental to our global understanding of gene therapy and have significantly contributed to the rapid expansion of the field. The use of adeno-associated viruses (AAVs) as vectors for gene transfer has successfully led to therapeutic expression of coagulation factor IX (FIX) in severe haemophilia B patients. Expression of FIX has remained stable following a single administration of vector for up to 8 years at levels that are clinically relevant to reduce the incidence of spontaneous bleeds and have permitted a significant change in the disease management with reduction or elimination of the need for coagulation factor concentrates. These trials have also shed light on several concerns around AAV-mediated gene transfer such as the high prevalence of pre-existing immunity against the vector capsid as well as the elevation of liver transaminases that is associated with a loss of FIX transgene expression in some patients. However, this field is advancing very rapidly with the development of increasingly more efficient strategies to overcome some of these obstacles and importantly raise the possibility of a functional cure, which has been long sought after. This review overviews the evolution of gene therapy for haemophilia B over the last two decades.

Asunto(s)

Hemofilia B , Humanos , Hemofilia B/genética , Hemofilia B/terapia , Vectores Genéticos , Terapia Genética , Factor IX/genética , Factor IX/uso terapéutico , Factor IX/metabolismo , Hemorragia/tratamiento farmacológico , Dependovirus/genética , Dependovirus/metabolismo

RESUMEN

Spinal cord injury (SCI) is a disorder of the central nervous system resulting from various factors such as trauma, inflammation, tumors, and other etiologies. This condition leads to impairment in motor, sensory, and autonomic functions below the level of injury. Limitations of current therapeutic approaches prompt an investigation into therapeutic angiogenesis through persistent local expression of proangiogenic factors. Here, we investigated whether overexpression of adeno-associated virus (AAV)-mediated vascular endothelial growth factor A (VEGFA) in mouse SCI promoted locomotor function recovery, and whether the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway was mechanistically involved. Three weeks before SCI, AAV-VEGFA was injected at the T10 level to induce VEGFA overexpression. Neurofunctional, histological, and biochemical assessments were done to determine tissue damage and/or recovery of neuromuscular and behavioral impairments. Daily injections of the PI3K/Akt pathway inhibitor LY294002 were made to assess a possible mechanism. AAV-VEGFA overexpression dramatically improved locomotor function and ameliorated pathological injury caused by SCI. Improved motor-evoked potentials in hindlimbs and more spinal CD31-positive microvessels were observed in AAV-VEGFA-overexpressing mice. LY294002 reduced PI3K and Akt phosphorylation levels and attenuated AAV-VEGFA-related improvements. In conclusion, sustained local AAV-mediated VEGFA overexpression in spinal cord can significantly promote angiogenesis and ameliorate locomotor impairment after SCI in a contusion mouse model through activation of the PI3K/Akt signaling pathway.

Asunto(s)

Proteínas Proto-Oncogénicas c-akt , Traumatismos de la Médula Espinal , Ratones , Animales , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Fosfatidilinositol 3-Quinasa/uso terapéutico , Angiogénesis , Transducción de Señal , Médula Espinal/patología , Recuperación de la Función/fisiología

RESUMEN

The enzyme choline acetyltransferase (ChAT) synthesizes acetylcholine from acetyl-CoA and choline at the neuromuscular junction and at the nerve terminals of cholinergic neurons. Mutations in the ChAT gene (CHAT) result in a presynaptic congenital myasthenic syndrome (CMS) that often associates with life-threatening episodes of apnea. Knockout mice for Chat (Chat-/-) die at birth. To circumvent the lethality of this model, we crossed mutant mice possessing loxP sites flanking Chat exons 4 and 5 with mice that expressed Cre-ERT2. Injection of tamoxifen (Tx) at postnatal (P) day 11 in these mice induced downregulation of Chat, autonomic failure, weakness, and death. However, a proportion of Chatflox/flox-Cre-ERT2 mice receiving at birth an intracerebroventricular injection of 2 × 1013 vg/kg adeno-associated virus type 9 (AAV9) carrying human CHAT (AAV9-CHAT) survived a subsequent Tx injection and lived to adulthood without showing signs of weakness. Likewise, injection of AA9-CHAT by intracisternal injection at P28 after the onset of weakness also resulted in survival to adulthood. The expression of Chat in spinal motor neurons of Chatflox/flox-Cre-ERT2 mice injected with Tx was markedly reduced, but AAV-injected mice showed a robust recovery of ChAT expression, which was mainly translated by the human CHAT RNA. The biodistribution of the viral genome was widespread but maximal in the spinal cord and brain of AAV-injected mice. No significant histopathological changes were observed in the brain, liver, and heart of AAV-injected mice after 1 year follow-up. Thus, AAV9-mediated gene therapy may provide an effective and safe treatment for patients severely affected with CHAT-CMS.

Asunto(s)

Colina O-Acetiltransferasa , Dependovirus , Ratones , Humanos , Animales , Colina O-Acetiltransferasa/genética , Colina O-Acetiltransferasa/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Distribución Tisular , Ratones Noqueados , Terapia Genética