Molecular Therapy. Methods & Clinical Development最新文献_第10页

Monitoring CAR T cell generation with a CD8-targeted lentiviral vector by single-cell transcriptomics. 通过单细胞转录组学监测靶向cd8的慢病毒载体的CAR - T细胞生成。

IF 4.7

Molecular Therapy. Methods & Clinical Development Pub Date : 2021-10-05 eCollection Date: 2021-12-10 DOI: 10.1016/j.omtm.2021.09.019

Filippos T Charitidis, Elham Adabi, Frederic B Thalheimer, Colin Clarke, Christian J Buchholz

{"title":"Monitoring CAR T cell generation with a CD8-targeted lentiviral vector by single-cell transcriptomics.","authors":"Filippos T Charitidis, Elham Adabi, Frederic B Thalheimer, Colin Clarke, Christian J Buchholz","doi":"10.1016/j.omtm.2021.09.019","DOIUrl":"https://doi.org/10.1016/j.omtm.2021.09.019","url":null,"abstract":"Quantifying gene expression in individual cells can substantially improve our understanding about complex genetically engineered cell products such as chimeric antigen receptor (CAR) T cells. Here we designed a single-cell RNA sequencing (scRNA-seq) approach to monitor the delivery of a CD19-CAR gene via lentiviral vectors (LVs), i.e., the conventional vesicular stomatitis virus (VSV)-LV and the CD8-targeted CD8-LV. LV-exposed human donor peripheral blood mononuclear cells (PBMCs) were evaluated for a panel of 400 immune response-related genes including LV-specific probes. The resulting data revealed a trimodal expression for the CAR and CD8A, demanding a careful distribution-based identification of CAR T cells and CD8+ lymphocytes in scRNA-seq analysis. The fraction of T cells expressing high CAR levels was in concordance with flow cytometry results. More than 97% of the cells hit by CD8-LV expressed the CD8A gene. Remarkably, the majority of the potential off-target cells were in fact on-target cells, resulting in a target cell selectivity of more than 99%. Beyond that, differential gene expression analysis revealed the upregulation of restriction factors in CAR-negative cells, thus explaining their protection from CAR gene transfer. In summary, we provide a workflow and subsetting approach for scRNA-seq enabling reliable distinction between transduced and untransduced cells during CAR T cell generation.","PeriodicalId":517056,"journal":{"name":"Molecular Therapy. Methods & Clinical Development","volume":" ","pages":"359-369"},"PeriodicalIF":4.7,"publicationDate":"2021-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/3c/ba/main.PMC8546366.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39673902","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 6

Safe and stable generation of induced pluripotent stem cells using doggybone DNA vectors. 利用狗骨DNA载体安全稳定地生成诱导多能干细胞。

IF 4.7

Molecular Therapy. Methods & Clinical Development Pub Date : 2021-10-05 eCollection Date: 2021-12-10 DOI: 10.1016/j.omtm.2021.09.018

Christopher D Thornton, Stuart Fielding, Kinga Karbowniczek, Alicia Roig-Merino, Alysha E Burrows, Lorna M FitzPatrick, Aseel Sharaireh, John P Tite, Sara E Mole, Richard P Harbottle, Lisa J Caproni, Tristan R McKay

{"title":"Safe and stable generation of induced pluripotent stem cells using doggybone DNA vectors.","authors":"Christopher D Thornton, Stuart Fielding, Kinga Karbowniczek, Alicia Roig-Merino, Alysha E Burrows, Lorna M FitzPatrick, Aseel Sharaireh, John P Tite, Sara E Mole, Richard P Harbottle, Lisa J Caproni, Tristan R McKay","doi":"10.1016/j.omtm.2021.09.018","DOIUrl":"https://doi.org/10.1016/j.omtm.2021.09.018","url":null,"abstract":"The application of induced pluripotent stem cells (iPSCs) in advanced therapies is increasing at pace, but concerns remain over their clinical safety profile. We report the first-ever application of doggybone DNA (dbDNA) vectors to generate human iPSCs. dbDNA vectors are closed-capped linear double-stranded DNA gene expression cassettes that contain no bacterial DNA and are amplified by a chemically defined, current good manufacturing practice (cGMP)-compliant methodology. We achieved comparable iPSC reprogramming efficiencies using transiently expressing dbDNA vectors with the same iPSC reprogramming coding sequences as the state-of-the-art OriP/EBNA1 episomal vectors but, crucially, in the absence of p53 shRNA repression. Moreover, persistent expression of EBNA1 from bacterially derived episomes resulted in stimulation of the interferon response, elevated DNA damage, and increased spontaneous differentiation. These cellular activities were diminished or absent in dbDNA-iPSCs, resulting in lines with a greater stability and safety potential for cell therapy.","PeriodicalId":517056,"journal":{"name":"Molecular Therapy. Methods & Clinical Development","volume":" ","pages":"348-358"},"PeriodicalIF":4.7,"publicationDate":"2021-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/a0/0d/main.PMC8546411.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39673901","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 5

Seven-year follow-up of durability and safety of AAV CNS gene therapy for a lysosomal storage disorder in a large animal. AAV CNS基因治疗大型动物溶酶体贮积症的持久性和安全性的7年随访。

IF 4.7

Molecular Therapy. Methods & Clinical Development Pub Date : 2021-10-05 eCollection Date: 2021-12-10 DOI: 10.1016/j.omtm.2021.09.017

Sara Marcó, Virginia Haurigot, Maria Luisa Jaén, Albert Ribera, Víctor Sánchez, Maria Molas, Miguel Garcia, Xavier León, Carles Roca, Xavier Sánchez, Joan Bertolin, Jennifer Pérez, Gemma Elias, Marc Navarro, Ana Carretero, Martí Pumarola, Anna Andaluz, Yvonne Espada, Sonia Añor, Fatima Bosch

{"title":"Seven-year follow-up of durability and safety of AAV CNS gene therapy for a lysosomal storage disorder in a large animal.","authors":"Sara Marcó, Virginia Haurigot, Maria Luisa Jaén, Albert Ribera, Víctor Sánchez, Maria Molas, Miguel Garcia, Xavier León, Carles Roca, Xavier Sánchez, Joan Bertolin, Jennifer Pérez, Gemma Elias, Marc Navarro, Ana Carretero, Martí Pumarola, Anna Andaluz, Yvonne Espada, Sonia Añor, Fatima Bosch","doi":"10.1016/j.omtm.2021.09.017","DOIUrl":"https://doi.org/10.1016/j.omtm.2021.09.017","url":null,"abstract":"Delivery of adeno-associated viral vectors (AAVs) to cerebrospinal fluid (CSF) has emerged as a promising approach to achieve widespread transduction of the central nervous system (CNS) and peripheral nervous system (PNS), with direct applicability to the treatment of a wide range of neurological diseases, particularly lysosomal storage diseases. Although studies in small animal models have provided proof of concept and experiments in large animals demonstrated feasibility in bigger brains, there is not much information on long-term safety or durability of the effect. Here, we report a 7-year study in healthy beagle dogs after intra-CSF delivery of a single, clinically relevant dose (2 × 1013 vg/dog) of AAV9 vectors carrying the canine sulfamidase, the enzyme deficient in mucopolysaccharidosis type IIIA. Periodic monitoring of CSF and blood, clinical and neurological evaluations, and magnetic resonance and ultrasound imaging of target organs demonstrated no toxicity related to treatment. AAV9-mediated gene transfer resulted in detection of sulfamidase activity in CSF throughout the study. Analysis at tissue level showed widespread sulfamidase expression and activity in the absence of histological findings in any region of encephalon, spinal cord, or dorsal root ganglia. Altogether, these results provide proof of durability of expression and long-term safety for intra-CSF delivery of AAV-based gene transfer vectors encoding therapeutic proteins to the CNS.","PeriodicalId":517056,"journal":{"name":"Molecular Therapy. Methods & Clinical Development","volume":" ","pages":"370-389"},"PeriodicalIF":4.7,"publicationDate":"2021-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8550992/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39698858","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 14

SAG therapy restores bone growth and reduces enchondroma incidence in a model of skeletal chondrodysplasias caused by Ihh deficiency. 在Ihh缺乏症引起的骨骼软骨发育不良模型中，SAG治疗可恢复骨生长并减少内生软骨瘤的发生率。

IF 4.7

Molecular Therapy. Methods & Clinical Development Pub Date : 2021-10-01 eCollection Date: 2021-12-10 DOI: 10.1016/j.omtm.2021.09.015

Xinhua Li, Shuting Yang, Zahra Chinipardaz, Eiki Koyama, Shuying Yang

{"title":"SAG therapy restores bone growth and reduces enchondroma incidence in a model of skeletal chondrodysplasias caused by Ihh deficiency.","authors":"Xinhua Li, Shuting Yang, Zahra Chinipardaz, Eiki Koyama, Shuying Yang","doi":"10.1016/j.omtm.2021.09.015","DOIUrl":"https://doi.org/10.1016/j.omtm.2021.09.015","url":null,"abstract":"Inactivation mutations in the Indian hedgehog (Ihh) gene in humans cause numerous skeletal chondrodysplasias, including acrocapitofemoral dysplasia, brachydactyly type A1, and human short stature. The lack of an appropriate human-relevant model to accurately represent these chondrodysplasias has hampered the identification of clinically effective treatments. Here, we established a mouse model of human skeletal dysplasia induced by Ihh gene mutations via ablation of Ihh in Aggrecan-positive (Acan+) cells using Aggrecan (Acan)-creERT transgenic mice. Smoothen agonist (SAG) promoted Hh activity and rescued chondrocyte proliferation and differentiation by stimulating smoothened trafficking to the cilium in Ihh-silenced cells. SAG treatment corrected mouse stature and significantly decreased mortality without evidence of toxicity. Moreover, Ihh ablation in Acan+ cells produced enchondroma-like tissues near the growth plates that were significantly reduced by SAG treatment. These results demonstrated that SAG effectively treats skeletal dysplasia caused by Ihh gene mutations in a mouse model, suggesting that SAG may represent a potential drug for the treatment of these diseases and/or enchondromas.","PeriodicalId":517056,"journal":{"name":"Molecular Therapy. Methods & Clinical Development","volume":" ","pages":"461-475"},"PeriodicalIF":4.7,"publicationDate":"2021-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/d2/a1/main.PMC8591400.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39768762","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 3

Lentiviral and adeno-associated vectors efficiently transduce mouse T lymphocytes when targeted to murine CD8. 慢病毒和腺相关载体在靶向小鼠CD8时有效地转导小鼠T淋巴细胞。

IF 4.7

Molecular Therapy. Methods & Clinical Development Pub Date : 2021-10-01 eCollection Date: 2021-12-10 DOI: 10.1016/j.omtm.2021.09.014

Alexander Michels, Annika M Frank, Dorothee M Günther, Mehryad Mataei, Kathleen Börner, Dirk Grimm, Jessica Hartmann, Christian J Buchholz

{"title":"Lentiviral and adeno-associated vectors efficiently transduce mouse T lymphocytes when targeted to murine CD8.","authors":"Alexander Michels, Annika M Frank, Dorothee M Günther, Mehryad Mataei, Kathleen Börner, Dirk Grimm, Jessica Hartmann, Christian J Buchholz","doi":"10.1016/j.omtm.2021.09.014","DOIUrl":"https://doi.org/10.1016/j.omtm.2021.09.014","url":null,"abstract":"Preclinical studies on gene delivery into mouse lymphocytes are often hampered by insufficient activity of lentiviral (LV) and adeno-associated vectors (AAVs) as well as missing tools for cell type selectivity when considering in vivo gene therapy. Here, we selected designed ankyrin repeat proteins (DARPins) binding to murine CD8. The top-performing DARPin was displayed as targeting ligand on both vector systems. When used on engineered measles virus (MV) glycoproteins, the resulting mCD8-LV transduced CD8+ mouse lymphocytes with near-absolute (>99%) selectivity. Despite its lower functional titer, mCD8-LV achieved 4-fold higher gene delivery to CD8+ cells than conventional VSV-LV when added to whole mouse blood. Addition of mCD8-LV encoding a chimeric antigen receptor (CAR) specific for mouse CD19 to splenocytes resulted in elimination of B lymphocytes and lymphoma cells. For display on AAV, the DARPin was inserted into the GH2-GH3 loop of the AAV2 capsid protein VP1, resulting in a DARPin-targeted AAV we termed DART-AAV. Stocks of mCD8-AAV contained similar genome copies as AAV2 but were >20-fold more active in gene delivery in mouse splenocytes, while exhibiting >99% specificity for CD8+ cells. These results suggest that receptor targeting can overcome blocks in transduction of mouse splenocytes.","PeriodicalId":517056,"journal":{"name":"Molecular Therapy. Methods & Clinical Development","volume":" ","pages":"334-347"},"PeriodicalIF":4.7,"publicationDate":"2021-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/fa/ea/main.PMC8531454.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39673900","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 9

GJB2 gene therapy and conditional deletion reveal developmental stage-dependent effects on inner ear structure and function. GJB2基因治疗和条件缺失揭示了发育阶段依赖性内耳结构和功能的影响。

IF 4.7

Molecular Therapy. Methods & Clinical Development Pub Date : 2021-10-01 eCollection Date: 2021-12-10 DOI: 10.1016/j.omtm.2021.09.009

Jingying Guo, Xiaobo Ma, Jennifer M Skidmore, Jelka Cimerman, Diane M Prieskorn, Lisa A Beyer, Donald L Swiderski, David F Dolan, Donna M Martin, Yehoash Raphael

{"title":"GJB2 gene therapy and conditional deletion reveal developmental stage-dependent effects on inner ear structure and function.","authors":"Jingying Guo, Xiaobo Ma, Jennifer M Skidmore, Jelka Cimerman, Diane M Prieskorn, Lisa A Beyer, Donald L Swiderski, David F Dolan, Donna M Martin, Yehoash Raphael","doi":"10.1016/j.omtm.2021.09.009","DOIUrl":"https://doi.org/10.1016/j.omtm.2021.09.009","url":null,"abstract":"Pathogenic variants in GJB2, the gene encoding connexin 26, are the most common cause of autosomal-recessive hereditary deafness. Despite this high prevalence, pathogenic mechanisms leading to GJB2-related deafness are not well understood, and cures are absent. Humans with GJB2-related deafness retain at least some auditory hair cells and neurons, and their deafness is usually stable. In contrast, mice with conditional loss of Gjb2 in supporting cells exhibit extensive loss of hair cells and neurons and rapidly progress to profound deafness, precluding the application of therapies that require intact cochlear cells. In an attempt to design a less severe Gjb2 animal model, we generated mice with inducible Sox10iCre ERT2 -mediated loss of Gjb2. Tamoxifen injection led to reduced connexin 26 expression and impaired function, but cochlear hair cells and neurons survived for 2 months, allowing phenotypic rescue attempts within this time. AAV-mediated gene transfer of GJB2 in mature mutant ears did not demonstrate threshold improvement and in some animals exacerbated hearing loss and resulted in hair cell loss. We conclude that Sox10iCre ERT2 ;Gjb2 flox/flox mice are valuable for studying the biology of connexin 26 in the cochlea. In particular, these mice may be useful for evaluating gene therapy vectors and development of therapies for GJB2-related deafness.","PeriodicalId":517056,"journal":{"name":"Molecular Therapy. Methods & Clinical Development","volume":" ","pages":"319-333"},"PeriodicalIF":4.7,"publicationDate":"2021-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/34/0f/main.PMC8531464.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39673899","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 10

Immunocompromised Cas9 transgenic mice for rapid in vivo assessment of host factors involved in highly pathogenic virus infection. 免疫功能受损的Cas9转基因小鼠用于体内快速评估参与高致病性病毒感染的宿主因子。

IF 4.7

Molecular Therapy. Methods & Clinical Development Pub Date : 2021-10-01 eCollection Date: 2021-12-10 DOI: 10.1016/j.omtm.2021.09.012

Nicole Collette, Pragyesh Dhungel, Sean J Lund, Jennifer L Schwedler, Edwin A Saada, Yooli K Light, Anupama Sinha, Joseph S Schoeniger, Oscar A Negrete

{"title":"Immunocompromised Cas9 transgenic mice for rapid in vivo assessment of host factors involved in highly pathogenic virus infection.","authors":"Nicole Collette, Pragyesh Dhungel, Sean J Lund, Jennifer L Schwedler, Edwin A Saada, Yooli K Light, Anupama Sinha, Joseph S Schoeniger, Oscar A Negrete","doi":"10.1016/j.omtm.2021.09.012","DOIUrl":"https://doi.org/10.1016/j.omtm.2021.09.012","url":null,"abstract":"Targeting host factors for anti-viral development offers several potential advantages over traditional countermeasures that include broad-spectrum activity and prevention of resistance. Characterization of host factors in animal models provides strong evidence of their involvement in disease pathogenesis, but the feasibility of performing high-throughput in vivo analyses on lists of genes is problematic. To begin addressing the challenges of screening candidate host factors in vivo, we combined advances in CRISPR-Cas9 genome editing with an immunocompromised mouse model used to study highly pathogenic viruses. Transgenic mice harboring a constitutively expressed Cas9 allele (Cas9 tg/tg ) with or without knockout of type I interferon receptors served to optimize in vivo delivery of CRISPR single-guide RNA (sgRNA) using Invivofectamine 3.0, a simple and easy-to-use lipid nanoparticle reagent. Invivofectamine 3.0-mediated liver-specific editing to remove activity of the critical Ebola virus host factor Niemann-Pick disease type C1 in an average of 74% of liver cells protected immunocompromised Cas9 tg/tg mice from lethal surrogate Ebola virus infection. We envision that immunocompromised Cas9 tg/tg mice combined with straightforward sgRNA in vivo delivery will enable efficient host factor loss-of-function screening in the liver and other organs to rapidly study their effects on viral pathogenesis and help initiate development of broad-spectrum, host-directed therapies against emerging pathogens.","PeriodicalId":517056,"journal":{"name":"Molecular Therapy. Methods & Clinical Development","volume":" ","pages":"286-295"},"PeriodicalIF":4.7,"publicationDate":"2021-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/bf/b4/main.PMC8526419.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39585542","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1

Novel TCR-like CAR-T cells targeting an HLA∗0201-restricted SSX2 epitope display strong activity against acute myeloid leukemia. 靶向HLA * 0201限制性SSX2表位的新型tcr样CAR-T细胞显示出对急性髓系白血病的强活性。

IF 4.7

Molecular Therapy. Methods & Clinical Development Pub Date : 2021-10-01 eCollection Date: 2021-12-10 DOI: 10.1016/j.omtm.2021.09.008

Scott Raskin, Stacey Van Pelt, Keri Toner, Preethi Bala Balakrishnan, Hema Dave, Catherine M Bollard, Eric Yvon

{"title":"Novel TCR-like CAR-T cells targeting an HLA∗0201-restricted SSX2 epitope display strong activity against acute myeloid leukemia.","authors":"Scott Raskin, Stacey Van Pelt, Keri Toner, Preethi Bala Balakrishnan, Hema Dave, Catherine M Bollard, Eric Yvon","doi":"10.1016/j.omtm.2021.09.008","DOIUrl":"https://doi.org/10.1016/j.omtm.2021.09.008","url":null,"abstract":"The synovial sarcoma X breakpoint 2 (SSX2) belongs to a multigene family of cancer-testis antigens and can be found overexpressed in multiple malignancies. Its restricted expression in immune-privileged normal tissues suggest that SSX2 may be a relevant target antigen for chimeric antigen receptor (CAR) therapy. We have developed a T cell receptor (TCR)-like antibody (Fab/3) that binds SSX2 peptide 41-49 (KASEKIFYV) in the context of HLA-A∗-0201. The sequence of Fab/3 was utilized to engineer a CAR with the CD3 zeta intra-cellular domain along with either a CD28 or 4-1BB costimulatory endodomain. Human T cells from HLA-A2+ donors were transduced to mediate anti-tumor activity against acute myeloid leukemia (AML) tumor cells. Upon challenge with HLA-A2+/SSX2+ AML tumor cells, CAR-expressing T cells released interferon-γ and eliminated the tumor cells in a long-term co-culture assay. Using the HLA-A2+ T2 cell line, we demonstrated a strong specificity of the single-chain variable fragment (scFv) for SSX2 p41-49 and the closely related SSX3 p41-49, with no response against the others SSX-homologous peptides or unrelated homologous peptides. Since SSX3 has not been observed in tumor cells and expression cannot be induced by pharmacological intervention, SSX241-49 represents an attractive target for CAR-based cellular therapy to treat multiple types of cancer.","PeriodicalId":517056,"journal":{"name":"Molecular Therapy. Methods & Clinical Development","volume":" ","pages":"296-306"},"PeriodicalIF":4.7,"publicationDate":"2021-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/e8/d6/main.PMC8526777.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39585543","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 9

Optimizing AAV2/6 microglial targeting identified enhanced efficiency in the photoreceptor degenerative environment. 优化AAV2/6小胶质靶向在光感受器退行性环境中提高了效率。

IF 4.7

Molecular Therapy. Methods & Clinical Development Pub Date : 2021-09-14 eCollection Date: 2021-12-10 DOI: 10.1016/j.omtm.2021.09.006

Margaret E Maes, Gabriele M Wögenstein, Gloria Colombo, Raquel Casado-Polanco, Sandra Siegert

{"title":"Optimizing AAV2/6 microglial targeting identified enhanced efficiency in the photoreceptor degenerative environment.","authors":"Margaret E Maes, Gabriele M Wögenstein, Gloria Colombo, Raquel Casado-Polanco, Sandra Siegert","doi":"10.1016/j.omtm.2021.09.006","DOIUrl":"https://doi.org/10.1016/j.omtm.2021.09.006","url":null,"abstract":"Adeno-associated viruses (AAVs) are widely used to deliver genetic material in vivo to distinct cell types such as neurons or glial cells, allowing for targeted manipulation. Transduction of microglia is mostly excluded from this strategy, likely due to the cells' heterogeneous state upon environmental changes, which makes AAV design challenging. Here, we established the retina as a model system for microglial AAV validation and optimization. First, we show that AAV2/6 transduced microglia in both synaptic layers, where layer preference corresponds to the intravitreal or subretinal delivery method. Surprisingly, we observed significantly enhanced microglial transduction during photoreceptor degeneration. Thus, we modified the AAV6 capsid to reduce heparin binding by introducing four point mutations (K531E, R576Q, K493S, and K459S), resulting in increased microglial transduction in the outer plexiform layer. Finally, to improve microglial-specific transduction, we validated a Cre-dependent transgene delivery cassette for use in combination with the Cx3cr1 CreERT2 mouse line. Together, our results provide a foundation for future studies optimizing AAV-mediated microglia transduction and highlight that environmental conditions influence microglial transduction efficiency.","PeriodicalId":517056,"journal":{"name":"Molecular Therapy. Methods & Clinical Development","volume":" ","pages":"210-224"},"PeriodicalIF":4.7,"publicationDate":"2021-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8516996/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39570033","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1

Evaluation of two in vitro assays for tumorigenicity assessment of CRISPR-Cas9 genome-edited cells. 两种体外检测CRISPR-Cas9基因组编辑细胞致瘤性的评价。

IF 4.7

Molecular Therapy. Methods & Clinical Development Pub Date : 2021-09-10 eCollection Date: 2021-12-10 DOI: 10.1016/j.omtm.2021.09.004

Myriam Lemmens, Benoit Fischer, Michael Zogg, Lindsey Rodrigues, Grainne Kerr, Alberto Del Rio-Espinola, Fanny Schaeffer, Danilo Maddalo, Valerie Dubost, Alessandro Piaia, Arne Mueller, Ulla Plappert-Helbig, Ulrike Naumann, Jasmin Haegele, Alex Odermatt, Hans-Jörg Martus, Silvana Libertini

{"title":"Evaluation of two in vitro assays for tumorigenicity assessment of CRISPR-Cas9 genome-edited cells.","authors":"Myriam Lemmens, Benoit Fischer, Michael Zogg, Lindsey Rodrigues, Grainne Kerr, Alberto Del Rio-Espinola, Fanny Schaeffer, Danilo Maddalo, Valerie Dubost, Alessandro Piaia, Arne Mueller, Ulla Plappert-Helbig, Ulrike Naumann, Jasmin Haegele, Alex Odermatt, Hans-Jörg Martus, Silvana Libertini","doi":"10.1016/j.omtm.2021.09.004","DOIUrl":"https://doi.org/10.1016/j.omtm.2021.09.004","url":null,"abstract":"Off-target editing is one of the main safety concerns for the use of CRISPR-Cas9 genome editing in gene therapy. These unwanted modifications could lead to malignant transformation, which renders tumorigenicity assessment of gene therapy products indispensable. In this study, we established two in vitro transformation assays, the soft agar colony-forming assay (SACF) and the growth in low attachment assay (GILA) as alternative methods for tumorigenicity evaluation of genome-edited cells. Using a CRISPR-Cas9-based approach to transform immortalized MCF10A cells, we identified PTPN12, a known tumor suppressor, as a valid positive control in GILA and SACF. Next, we measured the limit of detection for both assays and proved that SACF is more sensitive than GILA (0.8% versus 3.1% transformed cells). We further validated SACF and GILA by identifying a set of positive and negative controls and by testing the suitability of another cell line (THLE-2). Moreover, in contrast to SACF and GILA, an in vivo tumorigenicity study failed to detect the known tumorigenic potential of PTPN12 deletion, demonstrating the relevance of GILA and SACF in tumorigenicity testing. In conclusion, SACF and GILA are both attractive and valuable additions to preclinical safety assessment of gene therapy products.","PeriodicalId":517056,"journal":{"name":"Molecular Therapy. Methods & Clinical Development","volume":" ","pages":"241-253"},"PeriodicalIF":4.7,"publicationDate":"2021-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.omtm.2021.09.004","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39564698","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 4