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  • Effective multi-lineage engraftment in a mouse model of Fanconi anemia using non-genotoxic antibody-based conditioning
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2020-02-08
    Meera A. Srikanthan; Olivier Humbert; Kevin G. Haworth; Christina Ironside; Yogendra S. Rajawat; Bruce R. Blazar; Rahul Palchaudhuri; Anthony E. Boitano; Michael P. Cooke; David T. Scadden; Hans-Peter Kiem

    Conditioning chemotherapy is used to deplete hematopoietic stem cells in the recipient’s marrow, facilitating donor cell engraftment. While effective, a major issue with chemotherapy is the systemic genotoxicity that increases the risk of secondary malignancies. Antibody conjugates targeting hematopoietic cells is an emerging non-genotoxic method of opening the marrow niche and promoting engraftment of transplanted cells while maintaining intact marrow cellularity. Specifically, this platform would be useful in diseases associated with DNA damage or cancer predisposition such as dyskeratosis congenita, Schwachman-Diamond syndrome, and Fanconi anemia (FA). Our approach utilizes antibody-drug conjugates as an alternative conditioning regimen in an FA mouse model of autologous transplantation. Antibodies targeting either CD45 or CD117 were conjugated to saporin (SAP), a ribosomal toxin. Fanca knockout mice were conditioned with either CD45-SAP or CD117-SAP prior to receiving whole marrow from a heterozygous healthy donor. Bone marrow and peripheral blood analysis revealed equivalent levels of donor engraftment, with minimal toxicity in ADC-treated groups as compared to cyclophosphamide-treated controls. Our findings suggest ADCs may be an effective conditioning strategy in stem cell transplantation not only for diseases where traditional chemotherapy is not tolerated, but also more broadly for the field of blood and marrow transplantation.

  • A single “all-in-one” helper-dependent adenovirus to deliver donor DNA and CRISPR/Cas9 for efficient homology-directed repair
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2020-02-04
    Donna J. Palmer; Dustin L. Turner; Philip Ng

    In this study we developed a single helper-dependent adenovirus to deliver all of the components (donor DNA, Cas9 and gRNA) needed to achieve high efficiency gene targeting and homology-directed repair in transduced cells. We show that these “all-in-one” HDAds are up to 117-fold more efficient at gene targeting than donor HDAds that do not express CRISPR/Cas9 in human iPSCs. The vast majority (>90%) of targeted recombinants had only one allele targeted and this was accompanied by high frequency indel formation in the non-targeted allele at the site of Cas9 cleavage. These indels varied in size and nature and included large deletions of ∼8 kb. The remaining minority of recombinants had both alleles targeted (so-called bi-allelic targeting). These all-in-one HDAds represent an important platform for accomplishing and expanding the utility of homology-directed repair, especially for difficult-to-transfect cells and for in vivo applications.

  • Development and Preclinical Evaluation of an Integrase Defective Lentiviral Vector Vaccine Expressing the HIVACAT T Cell Immunogen in Mice
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2020-02-04
    Alessandra Gallinaro; Martina Borghi; Maria Franca Pirillo; Serena Cecchetti; Roberta Bona; Andrea Canitano; Zuleika Michelini; Antonio Di Virgilio; Alex Olvera; Christian Brander; Donatella Negri; Andrea Cara

    Cellular immune responses play a fundamental role in controlling viral replication and AIDS progression in HIV-infected subjects and in SIV-infected macaques. Integrase Defective Lentiviral Vector (IDLV) represents a promising vaccine candidate, inducing functional and durable immune responses in mice and non-human primates. Here, we designed HIV and SIV-based IDLVs to express the HIVACAT T cell immunogen (HTI), a mosaic antigen designed to cover vulnerable sites in HIV-1 Gag, Pol, Vif and Nef. We observed that HTI expression during lentiviral vector production interfered profoundly with IDLV particles release, due to sequestration of both HIV- and SIV-Gag proteins in the cytoplasm of the vector producing cells. However, modifications in IDLV design and vector production procedures greatly improved recovery of both HIV- and SIV-based IDLV-HTI. Immunization experiments in BALB/c mice showed that both IDLVs elicited HTI-specific T cell responses. However, immunization with HIV-based IDLV elicited also a T cell response towards exogenous HIV proteins in IDLV particles, suggesting that SIV-based IDLV may be a preferable platform to assess the induction of transgene-specific immune responses against rationally designed HIV structural antigens. These data support the further evaluation of IDLV as an effective platform of T cell immunogens for the development of an effective HIV-vaccine.

  • A High-Throughput Method for Characterizing Novel Chimeric Antigen Receptors in Jurkat Cells
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2020-01-31
    Darin Bloemberg; Tina Nguyen; Susanne MacLean; Ahmed Zafer; Christine Gadoury; Komal Gurnani; Anindita Chattopadhyay; Josée Ash; Julie Lippens; Doreen Harcus; Martine Pagé; Annie Fortin; Robert A. Pon; Rénald Gilbert; Anne Marcil; Risini D. Weeratna; Scott McComb

    Chimeric antigen receptor (CAR) development involves extensive empirical characterization of antigen binding domain (ABD)/CAR constructs for clinical suitability. Here, we present a cost-efficient and rapid method for evaluating CARs in human Jurkat T cells. Employing a modular CAR plasmid, a highly efficient ABD cloning strategy, plasmid electroporation, short-term co-culture, and flow cytometric detection of CD69, this assay (referred to as CAR-J) evaluates sensitivity and specificity for ABDs. Assessing 16 novel anti-CD22 single chain variable fragments derived from mouse monoclonal antibodies, CAR-J stratified constructs by response magnitude to CD22-expressing target cells. We also characterized 5 novel anti-EGFRvIII CARs for preclinical development, identifying candidates with varying tonic and target-specific activation characteristics. When evaluated in primary human T cells, tonic/auto-activating (without target cells) EGFRvIII-CARs induced target-independent proliferation, differentiation towards an effector phenotype, elevated activity against EGFRvIII-negative cells, and progressive loss of target-specific response upon in vitro re-challenge. These EGFRvIII CAR-T cells also showed anti-tumour activity in xenografted mice. In summary, CAR-J represents a straightforward method for high-throughput assessment of CAR constructs as genuine cell-associated antigen receptors that is particularly useful for generating large specificity datasets as well as potential downstream CAR optimization.

  • The mRNA-binding protein IGF2BP1 restores fetal hemoglobin in cultured erythroid cells from patients with β-hemoglobin disorders
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2020-01-31
    Christopher B. Chambers; Jeffrey Gross; Katherine Pratt; Xiang Guo; Colleen Byrnes; Y. Terry Lee; Donald Lavelle; Ann Dean; Jeffery L. Miller; Andrew Wilber
  • Rapid delivery of nanobodies/VHHs into living cells via expressing in vitro transcribed mRNA
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2020-01-30
    Xuechen Zhou; Rui Hao; Chen Chen; Zhipeng Su; Linhong Zhao; Zhuojuan Luo; Wei Xie

    Intracellular antigens labelling and manipulation by antibodies has been a long-thought goal in the field of cell research and therapy. However, a central limitation for this application is that antibodies are not able to penetrate into the cytosol of living cells. Taking advantages of small sizes and unique structures of the single-domain antibodies, here we presented a novel approach to rapidly deliver the nanobody/VHH into living cells via introducing its coding mRNA, which was generated by in vitro transcription. We demonstrated that actin-green fluorescent proteins (GFP) and Golgi-GFP can be recognized by the anti-GFP nanobody/VHH, vimentin can be recognized by the anti-vimentin nanobody/VHH, and histone deacetylase 6 (HDAC6) can be recognized by the anti-HDAC6 nanobody/VHH respectively. We found that the anti-GFP nanobody-mCherry expressed via in vitro transcribed (IVT) mRNA can be detected in 3 hours and degraded in 48 hours after transfection, while the nanobody-mCherry expressed via pmCherry-N1/anti-GFP nanobody was not detected until 24 hours after transfection. As a result, it is effective to deliver nanobody through expressing the nanobody/VHH in living cells from its coding mRNA.

  • A Rapid and Sensitive Nucleic Acid Amplification Technique for Mycoplasma Screening of Cell Therapy Products
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2020-01-30
    Lisa Dreolini; Mark Cullen; Eric Yung; Lawrence Laird; John R. Webb; Brad H. Nelson; Kevin A. Hay; Miruna Balasundaram; Natasha Kekre; Robert A. Holt

    Mycoplasma spp. bacteria can infect cell cultures, posing a potential threat to recipients of cell therapy products. Conventional Mycoplasma testing methods are highly sensitive but typically require a minimum of 28 days to produce results. This delay is problematic if rapid results are needed to inform treatment decisions. Nucleic Acid Amplification Technique (NAT) methods have been gaining favor for Mycoplasma testing due to their speed and specificity, however, they must first be qualified as meeting or exceeding the sensitivity of the compendial method. We present here a NAT method for the detection of Mycoplasma which circumvents the need for live Mycoplasma spp. in the test procedure by instead being qualified using Mycoplasma spp. genomic DNA. We have demonstrated a lower limit of detection that exceeds the regulatory requirements set by Health Canada. This assay is now being used to screen clinical cell therapy products manufactured at our centre.

  • A DNA vaccine that encodes an antigen presenting cell-specific heterodimeric protein protects against cancer and influenza
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2020-01-25
    Ranveig Braathen; Heidi Cecilie Larsen Spång; Daniëla Maria Hinke; Jana Blazevski; Sonja Bobic; Even Fossum; Bjarne Bogen

    Immunogenicity of DNA vaccines can be increased by constructing the DNA in such a way that it encodes secreted homodimeric fusion proteins that target antigen presenting cells (APC). We have here developed novel APC-targeting vaccine molecules with an increased flexibility due to introduction of a heterodimerization motif. The heterodimeric proteins permit four different fusions within a single molecule, thus allowing expression of two different APC-targeting moieties and two different antigens. Two types of heterodimeric fusion proteins were developed that employed either the ACID/BASE- or the Barnase/Barstar-motifs, respectively. The ACID/BASE heterodimeric vaccines conferred protection against challenges with either influenza virus or tumor cells in separate preclinical models. The ACID/BASE-motif was flexible since a large number of different targeting moieties and antigens could be introduced with maintenance of specificity, antigenicity and secretion. APC-targeting ACID/BASE vaccines expressing two different antigens induced antibody and T cell responses against either of the two antigens. Heterodimeric ACID/BASE DNA vaccines were of approximately the same potency as previously reported homodimeric DNA vaccines. The flexibility and potency of the ACID/BASE format suggest that it could be a useful platform for DNA vaccines that encode APC-targeting fusion proteins.

  • Factors Impacting Efficacy of AAV-mediated CRISPR-based Genome Editing for Treatment of Choroidal Neovascularization.
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2020-01-23
    Sook Hyun Chung; Iris Natalie Mollhoff; Uyen Nguyen; Amy Nguyen; Natalie Stucka; Eric Tieu; Suman Manna; Ratheesh Kumar Maleppat; Pengfei Zhang; Emerald Lovece Nguyen; Jared Fong; Robert Zawadzki; Glenn Yiu

    Frequent injections of anti-vascular endothelial growth factor (anti-VEGF) agents are a clinical burden for patients with neovascular age-related macular degeneration (AMD). Genomic disruption of VEGF-A using adeno-associated viral (AAV) delivery of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 has the potential to permanently suppress aberrant angiogenesis, but the factors that determine the optimal efficacy are unknown. Here, we investigate two widely-used Cas9 endonucleases, SpCas9 and SaCas9, and evaluate the relative contribution of AAV-delivery efficiency and genome-editing rates in vivo to determine the mechanisms that drive successful CRISPR-based suppression of VEGF-A, using a mouse model of laser-induced choroidal neovascularization (CNV). We found that SpCas9 demonstrated higher genome editing rates, greater VEGF reduction, and more effective CNV suppression than SaCas9, despite similar AAV transduction efficiency between a dual-vector approach for SpCas9 and single-vector system for SaCas9 to deliver the Cas9 orthologs and single guide RNAs (gRNAs). Our results suggest that successful VEGF knock-down using AAV-mediated CRISPR systems may be determined more by the efficiency of genome editing rather than viral transduction, and that SpCas9 may be more effective than SaCas9 as a potential therapeutic strategy for CRISPR-based treatment of CNV in neovascular AMD.

  • Therapeutic Mesenchymal Stromal Cells for Immunotherapy and for Gene and Drug Delivery
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2020-01-22
    Graça Almeida-Porada; Anthony J. Atala; Christopher D. Porada

    Mesenchymal stromal cells (MSC) possess several fairly unique properties that, combined, make them ideally suited for cellular-based immunotherapy and as vehicles for gene and drug delivery for a wide range of diseases/disorders. Key among these are: 1) their relative ease of isolation from a variety of tissues; 2) the ability to be expanded in culture without a loss of functionality, a property that varies to some degree with tissue source; 3) they are relatively immune-inert, perhaps obviating the need for precise donor/recipient matching; 4) they possess potent immunomodulatory functions that can be tailored by so-called licensing in vitro and in vivo; 5) the efficiency with which they can be modified with viral-based vectors; and 6) their almost uncanny ability to selectively home to damaged tissues, tumors, and metastases following systemic administration. In this review, we summarize the latest research in the immunological properties of MSC, their use as immunomodulatory/anti-inflammatory agents, methods for licensing MSC to customize their immunological profile, and their use as vehicles for transferring both therapeutic genes in genetic disease and drugs/genes designed to destroy tumor cells.

  • Suprachoroidal and subretinal injections of AAV using transscleral microneedles for retinal gene delivery in nonhuman primates
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2020-01-21
    Glenn Yiu; Sook Hyun Chung; Iris N. Mollhoff; Uyen Tu Nguyen; Sara M. Thomasy; Jesse Yoo; Donna Taraborelli; Glenn Noronha

    Retinal gene therapy using adeno-associated viruses (AAV) is constrained by the mode of viral vector delivery. Intravitreal AAV injections are impeded by the internal limiting membrane barrier, while subretinal injections require invasive surgery and produces a limited region of therapeutic effect. In this study, we introduce a novel mode of ocular gene delivery in rhesus macaques using transscleral microneedles to inject AAV8 into the subretinal or suprachoroidal space – a potential space between the choroid and scleral wall of the eye. Using in vivo imaging, we found that suprachoroidal AAV8 produces diffuse, peripheral expression in retinal pigment epithelial (RPE) cells, but elicited local infiltration of inflammatory cells. Transscleral subretinal injection of AAV8 using microneedles leads to focal gene expression with transduction of RPE and photoreceptors, and minimal intraocular inflammation. In comparison, intravitreal AAV8 shows minimal transduction of retinal cells, but elicits greater systemic humoral immune responses. Our study introduces a novel mode of transscleral viral delivery that can be performed without vitreoretinal surgery, with focal or diffuse transgene expression patterns suitable for different applications. The decoupling of local and systemic immune responses reveals important insights into the immunological consequences of AAV delivery to different ocular compartments surrounding the blood-retinal barrier.

  • Successful Transduction with AAV Vectors After Selective Depletion of Anti-AAV Antibodies by Immunoadsorption.
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2020-01-21
    Alejandro Orlowski; Michael G. Katz; Sarah M. Gubara; Anthony S. Fargnoli; Kenneth M. Fish; Thomas Weber

    Gene Therapy with adeno-associated virus (AAV) based vectors shows great promise for the gene therapeutic treatment of a broad array of diseases. In fact, the treatment of genetic diseases with AAV vectors is currently the only in vivo gene therapy approach that is approved by the U.S. Food and Drug Administration (FDA). Unfortunately, pre-existing antibodies against AAV severely limit the patient population that can potentially benefit from AAV gene therapy, especially if the vector is delivered by intravenous injection. Here, we demonstrate that we can selectively deplete anti-AAV antibodies by hemapheresis combined with AAV9 particles coupled to Sepharose beads. In rats that underwent hemapheresis and immunoadsorption luciferase expression was dramatically increased in the hearts and fully restored in the livers of these rats. Importantly, our method can be readily adapted for the use in clinical AAV gene therapy.

  • Chromosome transplantation: a possible approach to treat human X-linked disorders.
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2020-01-21
    Marianna Paulis; Lucia Susani; Alessandra Castelli; Teruhiko Suzuki; Takahiko Hara; Letizia Straniero; Stefano Duga; Dario Strina; Stefano Mantero; Elena Caldana; Lucia Sergi Sergi; Anna Villa; Paolo Vezzoni
  • CAR T cell generation by piggyBac transposition from linear doggybone™ DNA vectors requires transposon DNA flanking regions.
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2020-01-16
    David C. Bishop; Lisa Caproni; Kavitha Gowrishankar; Michal Legiewicz; Kinga Karbowniczek; John Tite; David J. Gottlieb; Kenneth P. Micklethwaite

    CD19-specific chimeric antigen receptor (CAR19) T cells generated using viral vectors are an efficacious but costly treatment for B cell malignancies. The non-viral piggyBac transposon system provides a simple and inexpensive alternative for CAR19 T cell production. Until now, piggyBac has been plasmid-based, facilitating economical vector amplification in bacteria. However, amplified plasmids have several undesirable qualities for clinical translation, including bacterial genetic elements, antibiotic resistance genes, and the requirement for purification to remove endotoxin. Doggybones (dbDNA™) are linear, covalently closed, minimal DNA vectors that can be inexpensively produced enzymatically in vitro at large scale. Importantly, they lack the undesirable features of plasmids. We used dbDNA incorporating piggyBac to generate CAR19 T cells. Initially, expression of functional transposase was evident, but stable CAR expression did not occur. After excluding other causes, additional random DNA flanking the transposon within the dbDNA was introduced, promoting stable CAR expression comparable to that using plasmid components. Our findings demonstrate that dbDNA incorporating piggyBac can be used to generate CAR T cells, and indicate that there is a requirement for DNA flanking the piggyBac transposon to enable effective transposition. dbDNA may further reduce the cost and improve the safety of CAR T cell production with transposon systems.

  • Identification of Key Coagulation Activity Determining Elements in Canine factor VIII
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2020-01-15
    Jenni Firrman; Qizhao Wang; Wenman Wu; Biao Dong; Wenjing Cao; Andrea Rossi Moore; Sean Roberts; Barbara A. Konkle; Carol Miao; LinShu Liu; Dong Li; Weidong Xiao
  • Oncolytic viruses and the immune system: The dynamic duo
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2020-01-15
    Ana Lemos de Matos; Lina S. Franco; Grant McFadden

    Oncolytic viruses (OVs) constitute a new and promising immunotherapeutic approach towards cancer treatment. This therapy takes advantage of the natural propensity of most tumor cells to be infected by specific OVs. Besides the direct killing potential (oncolysis), what makes OVs administration attractive for the present cancer immunotherapeutic scenario is the capacity to induce two new overlapping, but distinct, immunities: anti-tumoral and anti-viral. OV infection and oncolysis naturally elicits both innate and adaptive immune responses (required for long term anti-tumoral immunity); at the same time, the viral infection prompts an anti-viral response. In this review, we discuss the dynamic interaction between OVs and the triggered responses of the immune system. The anti-OV immunological events that lead to viral clearance and the strategies to deal with such potential loss of the therapeutic virus are discussed. Additionally, we review the immune stimulatory actions induced by OVs through different inherent strategies, such as modulation of the tumor microenvironment, the role of immunogenic cell death, and the consequences of genetically modifying the OV by arming with therapeutic transgenes. An understanding of the balance between the OV-induced anti-tumoral vs anti-viral immunities will provide insight when choosing the appropriate virotherapy for any specific cancer.

  • Digital-PCR assays for precise quantification of CD19-CAR T cells after treatment with Axicabtagene ciloleucel (Axi-cel)
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2020-01-15
    Boris Fehse; Anita Badbaran; Carolina Berger; Tanja Sonntag; Kristoffer Riecken; Maria Geffken; Nicolaus Kröger; Francis A. Ayuk
  • Manufacturing Dendritic Cells for Immunotherapy: Monocyte Enrichment
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2020-01-15
    Emily L. Hopewell; Cheryl Cox

    Dendritic cells play a key role in activation of the immune system as potent antigen-presenting cells. This pivotal position, along with the ability to generate dendritic cells from monocytes, and ready uptake of antigen makes them an intriguing vehicle for immunotherapy for a variety of indications. Since the first reported trial using dendritic cells in 1995, they have been used in trials all over the world for a plethora of indications. Monocyte-derived dendritic cells are generated from whole blood or apheresis products by culturing enriched monocytes in the presence of IL-4 and GM-CSF. A variety of methods can be used for enrichment of monocytes for generation of clinical-grade dendritic cells and will be summarized here.

  • Long-term metabolic correction of phenylketonuria by AAV delivered humanized phenylalanine amino lyase
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2020-01-13
    Rui Tao; Lin Xiao; Lifang Zhou; Zhaoyue Zheng; Jie Long; Lixing Zhou; Minghai Tang; Biao Dong; Shaohua Yao

    Phenylketonuria (PKU) is an inherited metabolic dissorder caused by mutation within phenylalanine hydroxylase (PAH) gene. Loss-of-function of PAH leads to accumulation of phenylalanine in the blood/body of untreated patient, which damages the developing brain, causing severe mental retardation. Current gene therapy strategies based on adeno-associated vector (AAV) delivery of PAH gene were effective in male animals while had little long-term effects on blood hyperphenylalaninemia in females. Here, we designed a gene therapy strategy using AAV to deliver a humanized phenylalanine amino lyase (hPAL) in a liver specific manner. It was showed that PAL was active in lysing phenylalanine when it was expressed in mammalian cells. We produced a recombinant adeno-associated vector serotype 8 (AAV8) viral vector expressing the humanized PAL under the control of human antitrypsin (hAAT) promoter (AAV8-PAL). A single intravenous administration of AAV8-PAL caused long-term correction of hyperphenylalaninemia in both male and female PKU mice (strain Pahenu2). Besides, no obvious liver injury was observed throughout the treatment process. Thus, our results established that AAV-mediated liver delivery of PAL gene is a promising strategy in the treatment of PKU.

  • Human iPSC-derived neural crest stem cells exhibit low immunogenicity
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2020-01-13
    Vera J. Mehler; Chris J. Burns; Hans Stauss; Robert J. Francis; Melanie L. Moore

    Recent clinical trials are evaluating induced-pluripotent stem cells (iPSCs) as a cellular therapy in the field of regenerative medicine. The widespread clinical utility of iPSCs is expected to be realised using allogeneic cells that have undergone thorough safety evaluations, including assessment of their immunogenicity. IPSC-derived neural crest stem cells (NCSCs) have significant potential in regenerative medicine, however, their application in cellular therapy has not been widely studied to date and no reports on their potential immunogenicity have been published so far. In this study, we have assessed the expression of immune-related antigens in iPSC-NCSCs, including HLA class I and II and co-stimulatory molecules. To investigate functional immunogenicity, iPSC-NCSCs were used as stimulator cells in a one-way mixed lymphocyte reaction. In these experiments, iPSC-NCSCs did not stimulate detectable proliferation of CD3+ and CD3+CD8+ T cells or induce cytokine production. We show that this was not a result of any immunosuppressive features of iPSC-NCSCs but rather more consistent with their non-immunogenic molecular phenotype. These results are encouraging for the potential future use of iPSC-NCSCs as a cellular therapy.

  • Point mutations in retargeted gD eliminate the sensitivity of EGFR/EGFRvIII-targeted HSV to key neutralizing antibodies
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2020-01-13
    Ceren Tuzmen; Tina M. Cairns; Doina Atanasiu; Huan Lou; Wan Ting Saw; Bonnie L. Hall; Justus B. Cohen; Gary H. Cohen; Joseph C. Glorioso

    Effective oncolytic virotherapy may require systemic delivery, tumor targeting and resistance to virus neutralizing (VN) antibodies. Since HSV glycoprotein D is the viral attachment/entry protein and predominant VN target, we examined the impact of gD retargeting alone and in combination with alterations in dominant VN epitopes on virus susceptibility to VN antibodies. We compared the binding of a panel of anti-gD monoclonal antibodies (mAbs) that mimic antibody specificities in human HSV-immune sera to the purified ectodomains of wild-type and retargeted gD, revealing the retention of two prominent epitopes. Substitution of a key residue in each epitope, separately and together revealed that both substitutions (i) blocked retargeted gD recognition by mAbs to the respective epitopes, and, in combination, caused a global reduction in mAb binding; (ii) protected against fusion inhibition by VN mAbs reactive with each epitope in virus-free cell-cell fusion assays; and (iii) increased the resistance of retargeted HSV-1 to these VN mAbs. Although the combined modifications of retargeted gD allowed bona fide retargeting, incorporation into virions was partially compromised. Our results indicate that stacking of epitope mutations can additively block retargeted gD recognition by VN antibodies but also that improvements in gD incorporation into virus particles may be required.

  • Novel GAA variants and mosaicism in Pompe disease identified by extended analyses of patients with an incomplete DNA diagnosis
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2020-01-13
    Stijn L.M. in ’t Groen; Douglas Oliveira Soares de Faria; Alessandro Iuliano; Johanna M.P. van den Hout; Hannie Douben; Trijnie Dijkhuizen; David Cassiman; Peter Witters; Miguel-Ángel Barba Romero; Annelies de Klein; Galhana M. Somers-Bolman; Jasper J. Saris; Lies H. Hoefsloot; Ans T. van der Ploeg; Atze J. Bergsma; W.W.M. Pim Pijnappel

    Pompe disease is a metabolic disorder caused by a deficiency of the glycogen-hydrolyzing lysosomal enzyme acid α-glucosidase (GAA), which leads to progressive muscle wasting. This autosomal recessive disorder is the result of disease-associated variants located in the GAA gene. In the present study we performed extended molecular diagnostic analysis to identify novel disease-associated variants in six suspected Pompe patients from four different families for which conventional diagnostic assays were insufficient. Additional assays such as a generic splicing assay, minigene analysis, SNP array analysis and targeted Sanger sequencing allowed the identification of an exonic deletion, a promoter deletion, and a novel splicing variant located in the 5' UTR. Furthermore, we describe the diagnostic process for an infantile patient with an atypical phenotype, consisting of left ventricular hypertrophy but no signs of muscle weakness or motor problems. This lead to the identification of a genetic mosaicism for a very severe GAA variant caused by a segmental uniparental isodisomy (UPD). With this study we aim to emphasize the need for additional analyses to detect new disease-associated GAA variants and non-Mendelian genotypes in Pompe disease where conventional DNA diagnostic assays are insufficient.

  • Expression of Neprilysin in Skeletal Muscle by Ultrasound-Mediated Gene Transfer (Sonoporation) Reduces Amyloid Burden for Alzheimer’s Disease
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2020-01-10
    Yuanli Li; Yadi Wang; Jue Wang; Ka Yee Chong; Jingjing Xu; Zhaohui Liu; Chunlei Shan

    Amyloid β (Aβ) accumulation in the brain is considered to be one of the major pathological changes in the progression of Alzheimer’s disease (AD). Neprilysin (NEP) is a zinc metallopeptidase that efficiently degrades Aβ. However, conventional approaches for increasing NEP levels or inducing its activation via viral-vector gene delivery have been shown to be problematic due to complications involving secondary toxicity, immune responses, and/or low gene transfer efficiency. Thus, in the present study, a physical and tractable NEP gene delivery system via ultrasound (US) combined with microbubbles was developed for AD therapy. We introduced the plasmid, human neprilysin (hNEP), into skeletal muscle of six-month-old amyloid precursor protein/presenilin-1 (APP/PS1) AD mice. Interestingly, we found a significantly reduced Aâ burden in the brain at one month after the delivery of over-expressed hNEP into skeletal muscle. Moreover, hNEP-treated AD mice exhibited improved performance in the Morris water maze compared to that of untreated AD mice. In addition, there were no apparent injuries in the injected muscle or in the lungs or kidneys at one month after the delivery of hNEP into skeletal muscle. These findings suggest that introducing hNEP into skeletal muscle via US represents an effective and safe therapeutic strategy for ameliorating AD-like symptoms in APP/PS1 mice, which may have the potential for clinical applications in the future.

  • In vivo Myoblasts Tracking Using The Sodium Iodide Symporter Gene Expression In Dogs
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2020-01-09
    Isabel Punzón; David Mauduit; Bryan Holvoet; Jean-Laurent Thibaud; Pauline de Fornel; Christophe M. Deroose; Nicolas Blanchard-Gutton; Jean-Thomas Vilquin; Maurilio Sampaolesi; Inès Barthélémy; Stéphane Blot
  • CRISPR-Cpf1 activation of endogenous BMP4 gene for osteogenic differentiation of umbilical cord-derived mesenchymal stem cells
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2020-01-09
    Jaehoon Choi; Taegeun Bae; Ninj Byambasuren; Seong-Ho Park; Chris H. Jo; Dokyoung Kim; Junho K. Hur; Nathaniel S. Hwang
  • Immune response mechanisms against AAV vectors in animal models
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-12-25
    Ashley T. Martino; David M. Markusic

    Early preclinical studies in rodents and other species did not reveal that vector or transgene immunity would present a significant hurdle for sustained gene expression. While there was early evidence of a mild immune responses to AAV in preclinical studies, it was generally believed that these responses were too weak and transient to negatively impact sustained transduction. However, translation of the cumulative success in treating hemophilia B in rodents and dogs with an AAV2-F9 vector to human studies was not as successful. Despite significant progress in recent clinical trials for hemophilia, new immunotoxicities to AAV and transgene are emerging in humans that require better animal models to assess and overcome these responses. The animal models designed to address these immune complications have provided critical information to assess how vector dose, vector capsid processing, vector genome, difference in serotypes and variations in vector delivery route can impact immunity and to develop approaches for overcoming pre-existing immunity. Additionally, a comprehensive dissection of innate, adaptive and regulatory responses to AAV vectors in preclinical studies has provided a framework that can be utilized for development of immunomodulatory therapies to overcome or bypass immune responses and for developing strategic approaches towards engineering stealth AAV vectors that can circumvent immunity.

  • VEGFD protects retinal ganglion cells and, consequently, capillaries against excitotoxic injury
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-12-25
    Annabelle Schlüter; Bahar Aksan; Ricarda Diem; Richard Fairless; Daniela Mauceri

    In the central nervous system, neurons and the vasculature influence each other. While it is well described that a functional vascular system is trophic to neurons and that vascular damage contributes to neurodegeneration; the opposite scenario in which neural damage might impact the microvasculature is less defined. Here, using an in vivo excitotoxic approach in adult mice as a tool to cause specific damage to retinal ganglion cells, we detected a subsequent damage to endothelial cells in retinal capillaries. Further, we detected decreased expression of Vascular Endothelial Growth Factor D (VEGFD) in retinal ganglion cells. In vivo VEGFD supplementation via neuronal-specific viral-mediated expression or acute intravitreal delivery of the mature protein preserved the structural and functional integrity of retinal ganglion cells against excitotoxicity and, additionally spared endothelial cells from degeneration. Viral-mediated suppression of expression of the VEGFD-binding receptor VEGFR3 in retinal ganglion cells revealed that VEGFD exerts its protective capacity directly on retinal ganglion cells while protection of endothelial cells is the result of upheld neuronal integrity. These findings suggest that VEGFD supplementation might be a novel, clinically applicable approach for neuronal and vascular protection.

  • ISPD overexpression enhances Ribitol-induced glycosylation of α-dystroglycan in dystrophic FKRP mutant mice
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-12-24
    Marcela P. Cataldi; Anthony Blaeser; Peijuan Lu; Victoria Leroy; Qi Long Lu

    Dystroglycanopathy, a subgroup of muscular dystrophies, is characterized by hypoglycosylation of α-dystroglycan (α-DG), which reduces its laminin-binding activity to extracellular matrix proteins, causing progressive loss of muscle integrity and function. Mutations in the fukutin-related protein (FKRP) gene are the most common causes of dystroglycanopathy. FKRP transfers ribitol-5-phosphate to the O-mannosyl glycan on α-DG from substrate CDP-ribitol, which is synthesized by Isoprenoid synthase domain-containing protein (ISPD). We previously reported that oral administration of ribitol restores therapeutic levels of functional glycosylation of α-DG (F-α-DG) in a FKRP mutant mouse model. Here we examine the contribution of AAV-mediated overexpression of ISPD to the levels of CDP-ribitol and F-α-DG with and without ribitol supplementation in the disease model. ISPD overexpression alone and in combination with ribitol improves dystrophic phenotype. Furthermore, the combined approach of ribitol and ISPD acts synergistically increasing F-α-DG up to 40% of normal levels in cardiac muscle, and more than 20% in limb and diaphragm. The results suggest that low levels of substrate limit production of CDP-ribitol, and endogenous ISPD also become a limiting factor in presence of supraphysiological concentration of ribitol. Our data support further investigation of the regulatory pathway for enhancing efficacy of ribitol supplement to FKRP-related dystroglycanopathy.

  • T cell-derived iPSCs as a source of regenerated T cells: high frequency production of iPSC clones capable of generating potent cytotoxic T cells
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-12-24
    Seiji Nagano; Maeda Takuya; Hiroshi Ichise; Soki Kashima; Manami Ohtaka; Mahito Nakanishi; Toshio Kitawaki; Norimitsu Kadowaki; Akifumi Takaori-Kondo; Kyoko Masuda; Hiroshi Kawamoto

    Current adoptive T cell therapies conducted in an autologous setting are costly, time-consuming and depend on the quality of the patient’s T cells, thus it would be highly beneficial to develop an allogeneic strategy. To this aim, we have developed a method by which cytotoxic T lymphocytes (CTLs) are regenerated from induced pluripotent stem cells that are originally derived from T cells (T-iPSCs). In order to assess the feasibility of this strategy, we investigated the frequency of usable T-iPSC clones in terms of their T cell generating capability and TCR affinity. We first established eight clones of T-iPSCs bearing different MART-1-specific TCRs from a healthy volunteer. Whereas all clones were able to give rise to mature CTLs, cell yield varied greatly, and five clones were considered to be usable. TCR affinity in the regenerated CTLs showed a large variance among the eight clones, but functional avidities measured by cytotoxic activity were almost equivalent among three selected clones representing high, medium and low TCR affinity. In a total of 50 alloreactivity tests using five CTL clones versus ten target cells, alloreactivity was seen in only three cases. These findings collectively support the feasibility of this T-iPSC strategy.

  • Targeting antigen to the surface of extracellular vesicles improves the in vivo immunogenicity of human and non-human adenoviral vaccines in mice
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-12-24
    C.M. Bliss; A.J. Parsons; R. Nachbagauer; J.R. Hamilton; F. Cappuccini; M. Ulaszewska; J.P. Webber; A. Clayton; A.V.S. Hill; L. Coughlan
  • 7T MRI predicts amelioration of neurodegeneration in the brain after AAV gene therapy
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-12-24
    Heather L. Gray-Edwards; Anne S. Maguire; Nouha Salibi; Lauren E. Ellis; Taylor L. Voss; Elise B. Diffie; Jey Koehler; Ashley N. Randle; Amanda R. Taylor; Brandon L. Brunson; Thomas S. Denney; Ronald J. Beyers; Atoska S. Gentry; Amanda L. Gross; Ana R. Batista; Miguel Sena-Esteves; Douglas R. Martin

    GM1 gangliosidosis is a fatal neurodegenerative lysosomal storage disease that occurs most commonly in young children, with no effective treatment available. Long-term follow up of GM1 cats treated by bilateral thalamic and deep cerebellar nuclei injection of AAV mediated gene therapy has increased lifespan to 8 years of age, compared to an untreated lifespan of ∼8 months. Due to risks associated with cerebellar injection in humans, the lateral ventricle was tested as a replacement route to deliver an AAVrh8 vector expressing feline β-galactosidase (β-gal), the defective enzyme in GM1 gangliosidosis. Treatment via the thalamus and lateral ventricle corrected storage, myelination, astrogliosis and neuronal morphology in areas where β-gal was effectively delivered. Oligodendrocyte number increased, but only in areas where myelination was corrected. Reduced AAV and β-gal distribution were noted in the cerebellum with subsequent increases in storage, demyelination, astrogliosis and neuronal degeneration. These postmortem findings were correlated with endpoint MRI and MR spectroscopy. Compared to the moderate dose with which most cats were treated, a higher AAV dose produced superior survival, currently 6.5 years. Thus, MRI and MRS can predict therapeutic efficacy of AAV gene therapy and non-invasively monitor cellular events within the GM1 brain.

  • Lentiviral Vector Production Titre is not limited in HEK 293T by Induced Intracellular Innate Immunity
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-12-24
    Carolina B. Ferreira; Rebecca P. Sumner; Maria T. Rodriguez-Plata; Jane Rasaiyaah; Richard S. Milne; Adrian J. Thrasher; Waseem Qasim; Greg J. Towers

    Most gene therapy lentiviral vector (LV) production platforms employ HEK 293T cells expressing the oncogenic SV40 large T-antigen (TAg) that is thought to promote plasmid-mediated gene expression. Studies on other viral oncogenes suggest that TAg may also inhibit the intracellular autonomous innate immune system that triggers defensive antiviral responses upon detection of viral components by cytosolic sensors. Here we show that an innate response can be generated after HIV-1-derived LV transfection in HEK 293T cells, particularly by the transgene, yet, remarkably, this had no effect on LV titre. Further, overexpression of DNA sensing pathway components led to expression of inflammatory cytokine and interferon (IFN) stimulated genes but did not result in detectable IFN or CXCL10 and had no impact on LV titre. Exogenous IFNβ also did not affect LV production or transduction efficiency in primary T cells. Additionally, manipulation of TAg did not affect innate antiviral responses, but stable expression of TAg boosted vector production in HEK 293 cells. Our findings demonstrate a measure of innate immune competence in HEK 293T cells but, crucially, show that activation of inflammatory signalling is uncoupled from cytokine secretion in these cells. This provides new mechanistic insight into the unique suitability of HEK 293T cells for lentiviral vector manufacture.

  • Genetic engineering of T cells for immune tolerance
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-12-24
    David W. Scott

    Regulatory T cells (Tregs) play a role in the induction and maintenance of tolerance, as well as in modulating aberrant immune responses. While expanded Tregs have been used in clinical trials, they are polyclonal and the frequency of specific Tregs is very low. To overcome this issue, we have endeavored to “specify” Tregs by engineering them to express receptors that can recognize a given antigen, and applied this protocol in autoimmunity, hemophilia and allergy. Thus, we have used retroviral transduction of specific T-cell receptor, single chain Fv or antigen domains in regulatory T cells to achieve this goal. This review summarizes our steps to achieve the ultimate goal of modulating human diseases.

  • Intralingual and intrapleural AAV gene therapy prolongs survival in a SOD1 ALS mouse model
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-12-24
    Allison M. Keeler; Marina Zieger; Carson Semple; Logan Pucci; Alessandra Veinbachs; Robert H. Brown; Christian Mueller; Mai K. ElMallah

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that results in death from respiratory failure. No cure exists for this devastating disease, but therapy that directly targets the respiratory system has the potential to prolong survival and improve quality of life in some cases of ALS. The objective of this study is to enhance breathing and prolong survival by suppressing SOD1 expression in respiratory motor neurons using adeno-associated virus expressing an artificial microRNA targeting the SOD1 gene. AAV-miRSOD1 was injected in the tongue and intrapleural space of SOD1G93A mice and repetitive respiratory and behavioral measurements were performed until end stage. Robust silencing of SOD1 was observed in the diaphragm and tongue as well as systemically. Silencing of SOD1 prolonged survival by approximately 50 days, and delayed weight loss and limb weakness in treated animals compared to untreated controls. Histologically, there was preservation of the neuromuscular junctions in the diaphragm as well as number of axons in the phrenic and hypoglossal nerves. Although SOD1 suppression improved breathing and prolonged survival, it did not ameliorate the restrictive lung phenotype. Suppression of SOD1 expression in motor neurons that underlie respiratory function prolongs survival and enhances breathing until end stage in SOD1G93A ALS mice.

  • AAV-Mediated CRISPR/Cas9 Gene Editing in Murine Phenylketonuria
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-12-24
    Daelyn Y. Richards; Shelley R. Winn; Sandra Dudley; Sean Nygaard; Taylor L. Mighell; Markus Grompe; Cary O. Harding

    Phenylketonuria (PKU) due to recessively-inherited phenylalanine hydroxylase (PAH) deficiency results in hyperphenylalaninemia which is toxic to the central nervous system. Restriction of dietary phenylalanine intake remains the standard of PKU care and prevents the major neurologic manifestations of the disease, yet shortcomings of dietary therapy remain, including poor adherence to a difficult and unpalatable diet, an increased incidence of neuropsychiatric illness, and imperfect neurocognitive outcomes. Gene therapy for PKU is a promising novel approach to promote lifelong neurological protection while allowing unrestricted dietary phenylalanine intake. In this study, liver-tropic recombinant AAV2/8 vectors were used to deliver CRISPR/Cas9 machinery and facilitate correction of the Pahenu2 allele by homologous recombination. Additionally, a non-homologous end joining (NHEJ) inhibitor, vanillin, was co-administered with the viral drug to promote homology directed repair (HDR) with the AAV-provided repair template. This combinatorial drug administration allowed for lifelong, permanent correction of the Pahenu2 allele in a portion of treated hepatocytes of PKU mice, yielding partial restoration of liver PAH activity, substantial reduction of blood phenylalanine, and prevention of maternal PKU effects during breeding. This work reveals CRISPR/Cas9 gene editing is a promising tool for permanent PKU gene editing.

  • Urocortin 2 Gene Transfer Improves Glycemic Control and Reduces Retinopathy and Mortality in Murine Insulin Deficiency
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-12-14
    Mei Hua Gao; Dimosthenis Giamouridis; N Chin Lai; Tracy Guo; Bing Xia; Young Chul Kim; Viet Anh Nguyen Huu; Dorota Skowronska-Krawczyk; Louise Lantier; Raag Bhargava; H Kirk Hammond

    Type 1 diabetes affects 20 million patients worldwide. Insulin is the primary and commonly the sole therapy for type 1 diabetes. However, only a minority of patients attain the targeted glucose control and reduced adverse events. We tested urocortin 2 gene transfer as single-agent therapy for insulin deficiency using two mouse models. Urocortin 2 gene transfer reduced blood glucose for months after a single intravenous injection, through increased skeletal muscle insulin sensitivity, increased insulin release in response to glucose stimulation, and increased plasma insulin levels before and during euglycemic clamp. The combined increases in both insulin availability and sensitivity resulted in improved glycemic indices—events that were not anticipated in these insulin deficient models. In addition, urocortin 2 gene transfer reduced ocular manifestations of longstanding insulin deficiency such as vascular leak, and improved retinal function. Finally, mortality was reduced by urocortin 2 gene transfer. The mechanisms for these beneficial effects included increased activities of AMP-activated protein kinase and Akt in skeletal muscle, increased skeletal muscle glucose uptake, and increased insulin release. These data suggest that urocortin 2 gene transfer may be a viable therapy for new onset type 1 diabetes and might reduce insulin needs in later stage disease.

  • AAVrh10 vector corrects disease pathology in MPS IIIA mice and achieves widespread distribution of sulfamidase in the brain of large animals
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-12-10
    Michaël Hocquemiller, Kim M. Hemsley, Meghan L. Douglass, Sarah J. Tamang, Daniel Neumann, Barbara M. King, Helen Beard, Paul J. Trim, Leanne K. Winner, Adeline A. Lau, Marten F. Snel, Cathy Gomila, Jérôme Ausseil, Xin Mei, Laura Giersch, Mark Plavsic, Ralph Laufer

    Patients with mucopolysaccharidosis IIIA (MPS IIIA) lack the lysosomal enzyme sulfamidase (SGSH), which is responsible for the degradation of heparan sulfate (HS). Build-up of undegraded HS results in severe progressive neurodegeneration for which there is currently no treatment. The ability of the vector AAVrh.10-CAG-SGSH (LYS-SAF302) to correct disease pathology was evaluated in a mouse model for MPS IIIA. LYS-SAF302 was administered to 5-week-old MPS IIIA mice at three different doses (8.6E+08, 4.1E+10, and 9.0E+10 vg/animal) injected into the caudate putamen/striatum and thalamus. LYS-SAF302 was able to dose-dependently correct or significantly reduce HS storage, secondary accumulation of GM2 and GM3 gangliosides, ubiquitin-reactive axonal spheroid lesions, lysosomal expansion and neuroinflammation, at 12-weeks and 25-weeks post-dosing. To study SGSH distribution in the brain of large animals, LYS-SAF302 was injected into the subcortical white matter of dogs (1.0 or 2.0E+12 vg/animal) and cynomolgus monkeys (7.2E+11 vg/animal). Increases of SGSH enzyme activity of at least 20% above endogenous levels were detected in 78% (dogs 4 weeks after injection) and 97% (monkeys 6 weeks after injection) of the total brain volume. Taken together, these data validate intraparenchymal AAV administration as a promising method to achieve widespread enzyme distribution and correction of disease pathology in MPS IIIA.

  • Comparisons of infant and adult mice reveal age effects for liver depot gene therapy in Pompe disease
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-12-06
    Sang-oh Han, Songtao Li, Angela McCall, Benjamin Arnson, Jeffrey I. Everitt, Haoyue Zhang, Sarah P. Young, Mai K. ElMallah, Dwight D. Koeberl

    Pompe disease is caused by the deficiency of lysosomal acid α-glucosidase (GAA). It is expected that gene therapy to replace GAA with adeno-associated virus (AAV) vectors will be less effective early in life due to the rapid loss of vector genomes. AAV2/8-LSPhGAA (3x1010 vg/mouse) was administered to infant (2 week old) or adult (2 month old) GAA knockout mice. AAV vector transduction in adult mice significantly corrected GAA deficiency in the heart (p<0.0001), diaphragm (<0.01), and quadriceps (<0.001) for >50 weeks. However, in infant mice the same treatment only partially corrected GAA deficiency in heart (<0.05), diaphragm (<0.05), and quadriceps (<0.05). The clearance of glycogen was much more efficient in adult mice compared to infant mice. Improved wirehang test latency was observed for treated adults (<0.05), but not for infant mice. Abnormal ventilation was corrected in both infant and adult mice. Vector-treated female mice demonstrated functional improvement, despite a lower degree of biochemical correction compared with male mice. The relative vector dose for infants was approximately 3-fold higher than adults, when normalized to body weight at the time of vector administration. Given these data, the dose requirement to achieve similar efficacy will be higher for the treatment of young patients.

  • DNAJC14 ameliorates inner ear degeneration in the DFNB4 mouse model
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-11-30
    Hye Ji Choi, Hyun Jae Lee, Jin Young Choi, Ik Hyun Jeon, Byunghwa Noh, Sushil Devkota, Han-Woong Lee, Seong Kug Eo, Jae Young Choi, Min Goo Lee, Jinsei Jung

    The His723Arg (H723R) mutation in SLC26A4, encoding pendrin, is the most prevalent mutation in East Asia, resulting in DFNB4, an autosomal recessive type of genetic hearing loss. Although the main pathological mechanism of H723R was identified as a protein-folding defect in pendrin, there is still no curative treatment for associated hearing loss. Here, we show that H723R-pendrin expression and activity are rescued by activation of the chaperonin DNAJC14. In vitro, DNAJC14 was activated via Japanese encephalitis virus (JEV) inoculation, and toxin-attenuated JEV rescued the surface expression and anion exchange activity of H723R-pendrin. Human H723R-pendrin transgenic mice (hH723R Tg) were established in a mouse slc26a4 knock-out background, in which only hH723R-pendrin was expressed in the inner ear (Pax2-Cre dependent) to mimic human DFNB4 pathology. Crossing hH723R Tg with DNAJC14-overexpressing mice resulted in reduced cochlear hydrops and more preserved outer hair cells in the cochlea compared to those in hH723R Tg mice. Furthermore, the stria vascularis and spiral ligament were thicker and KCNJ10 expression was increased with DNAJC14 overexpression; however, hearing function and enlarged endolymphatic hydrops were not recovered. These results indicate that DNAJC14 overexpression ameliorates the cochlear degeneration caused by misfolded pendrin and might be a potential therapeutic target for DFNB4.

  • Optimizing CAR-T Cell Manufacturing Processes During Pivotal Clinical Trials
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-11-29
    Seshu Tyagarajan, Tom Spencer, Jonathan Smith

    Tisagenlecleucel is a CD19-specific chimeric antigen receptor (CAR)-T cell therapy approved for patients aged ≤25 years with relapsed/refractory B-cell precursor acute lymphoblastic leukemia (B-ALL) and adults with relapsed/refractory diffuse large B-cell lymphoma (DLBCL). The initial tisagenlecleucel manufacturing process technology was developed at an academic center and was subsequently transferred, optimized, validated, and scaled out to supply large global trials before commercialization. Tisagenlecleucel manufactured in 2 centralized facilities has been successfully used in global multicenter trials for B-ALL and DLBCL (>50 clinical centers in 12 countries). In this paper, we describe some of the continuous process improvements made to tisagenlecleucel manufacturing over time to meet global demand while maintaining and improving product quality. During early tisagenlecleucel clinical trials, process enhancements were made to address logistical challenges related to manufacturing for multicenter trials and to accommodate the variability observed in patient starting cellular material. These enhancements resulted in improvements in manufacturing capacity, process robustness, process success rates, and product quality, and led to reductions in throughput time. In summary, through continuous evaluation and improvements based on experience during global trials, a consistent and robust commercial manufacturing process for tisagenlecleucel has been developed, leading to improvements in manufacturing success versus the initial processes.

  • C3 transferase-expressing scAAV2 transduces ocular anterior segment tissues and lowers intraocular pressure in mouse and monkey
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-11-29
    Junkai Tan, Xizhen Wang, Suping Cai, Fen He, Daren Zhang, Dongkan Li, Xianjun Zhu, Liang Zhou, Ning Fan, Xuyang Liu

    Glaucoma is a lifelong disease with elevated intraocular pressure (IOP) as the main risk factor and reduction of IOP remains the major treatment for this disease. However, current IOP lowering therapies are far from being satisfactory. We have demonstrated the lentivirus-mediated exoenzyme C3 transferase (C3) expression in rat and monkey eyes induced relatively long-term IOP reduction. We now show that intracameral injection of self-complementary AAV2 containing a C3 gene into mouse and monkey eyes resulted in morphological changes in trabecular meshwork and IOP reduction. The vector transduced corneal endothelium and the C3 transgene expression, not vector itself, induced corneal edema as a result of actin associated endothelial barrier disruption. There was a positive (quadratic) correlation between measured IOP and grade of corneal edema. This is the first report of using an AAV to transduce the trabecular meshwork of monkeys with a gene capable of altering cellular structure and physiology, indicating a potential gene therapy for glaucoma.

  • Genome editing in patient iPSC efficiently corrects the most prevalent USH2A mutations and reveals intriguing mutant mRNA expression profiles
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-11-27
    Carla Sanjurjo-Soriano, Nejla Erkilic, David Baux, Daria Mamaeva, Christian P. Hamel, Isabelle Meunier, Anne-Françoise Roux, Vasiliki Kalatzis

    Inherited retinal dystrophies (IRDs) are characterized by progressive photoreceptor degeneration and vision loss. Usher syndrome (USH) is a syndromic IRD characterized by retinitis pigmentosa (RP) and hearing loss. USH is clinically and genetically heterogeneous, and the most prevalent causative gene is USH2A. USH2A mutations also account for a large number of isolated autosomal recessive RP (arRP) cases. This high prevalence is due to two recurrent USH2A mutations, c.2276G>T and c.2299delG. Due to the large size of the USH2A cDNA, gene augmentation therapy is inaccessible. However, CRISPR/Cas9-mediated genome editing is a viable alternative. We used eSpCas9 to successfully achieve seamless correction of the two most prevalent USH2A mutations in induced pluripotent stem cells (iPSCs) of patients with USH or arRP. Our results highlight features that promote high target efficacy and specificity of eSpCas9. Consistently, we did not identify any off-target mutagenesis in the corrected iPSC, which also retained pluripotency and genetic stability. Furthermore, analysis of USH2A expression unexpectedly identified aberrant mRNA levels associated with the c.2276G>T and c.2299delG mutations that were reverted following correction. Taken together, our efficient CRISPR/Cas9-mediated strategy for USH2A mutation correction brings hope for a potential treatment for USH and arRP patients.

    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-11-27
    Buel D. Rodgers, Yemeserach Bishaw, Denali Kagel, Julian N. Ramos, Joseph W. Maricelli

    Micro-dystrophin (μDys) gene therapeutics can improve striated muscle structure and function in different animal models of Duchenne muscular dystrophy. Most studies, however, used young mdx mice that lack a pronounced dystrophic phenotype, short treatment periods and limited muscle function tests. We therefore determined the relative efficacy of two previously described μDys gene therapeutics (rAAV6:μDysH3 and rAAV6:μDys5) in 6 m.o. mdx mice using a 6-month treatment regimen and forced exercise. Forelimb and hindlimb grip strength, metabolic rate (VO2 max), running efficiency (energy expenditure) and serum creatine kinase levels similarly improved in mdx mice treated with either vector. Both vectors produced nearly identical dose-responses in all assays. They also partially prevented the degenerative effects of repeated high-intensity exercise on muscle histology, although none of the metrics examined was restored to normal wild-type levels. Moreover, neither vector had any consistent effect on respiration while exercising. These data together suggest that although μDys gene therapy can improve isolated and systemic muscle function, it may be only partially effective when dystrophinopathies are advanced or when muscle structure is significantly challenged as with high-intensity exercise. This further suggests that restoring muscle function to near-normal levels will likely require ancillary or combinatorial treatments capable of enhancing muscle strength.

  • Nontoxic hematopoietic stem cell transplantation-based macrophage/microglia-mediated GDNF delivery for Parkinson’s disease
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-11-26
    Cang Chen, Michael J. Guderyon, Yang Li, Guo Ge, Anindita Bhattacharjee, Cori Ballard, Zhixu He, Eliezer Masliah, Robert A. Clark, Jason C. O'Connor, Senlin Li
  • Adeno-associated viral vectors in neuroscience research
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-11-26
    David L. Haggerty, Gregory G. Grecco, Kaitlin C. Reeves, Brady Atwood

    Adeno-associated viral vectors (AAVs) are increasingly useful preclinical tools in neuroscience research studies for interrogating cellular and neurocircuit function and mapping of brain connectivity. Clinically, AAVs are showing increasing promise as viable candidates for treating multiple neurological diseases. Here, we briefly review the utility of AAVs in mapping neurocircuits, manipulating neuronal function and gene expression, and activity labeling in preclinical research studies as well as AAV-based gene therapies for diseases of the nervous system. This review highlights the vast potential that AAVs have for transformative research and therapeutics in the neurosciences.

  • Production of Lentiviral Vectors Using Suspension Cells Grown in Serum-Free Media
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-11-26
    Matthew Bauler, Jessica K. Roberts, Chang-Chih Wu, Baochang Fan, Francesca Ferrara, Bon Ham Yip, Shiyong Diao, Young-In Kim, Jennifer Moore, Sheng Zhou, Matthew M. Wielgosz, Byoung Ryu, Robert E. Throm

    Lentiviral vectors are increasingly utilized in cell and gene therapy applications as they efficiently transduce target cells such as hematopoietic stem cells and T-cells. Large-scale production of current Good Manufacturing Practices-grade lentiviral vectors is limited because of the adherent, serum-dependent nature of HEK293T cells used in the manufacturing process. To optimize large-scale clinical-grade lentiviral vector production, we developed an improved production scheme by adapting HEK293T cells to grow in suspension using commercially available and chemically-defined serum-free media. Lentiviral vectors with titers equivalent to those of HEK293T cells were produced from SJ293TS cells using optimized transfection conditions which reduced the required amount of plasmid DNA by 50%. Furthermore, purification of SJ293TS-derived lentiviral vectors at 1 L yielded a recovery of 55 ± 14% (n=138) of transducing units in the starting material, more than a two-fold increase over historical yields from adherent HEK293T serum-dependent lentiviral vector preparations. SJ293TS cells were stable to produce lentiviral vectors over four months of continuous culture. SJ293TS-derived lentiviral vectors efficiently transduced primary hematopoietic stem cells and T-cells from healthy donors. Overall, our SJ293TS cell line enables high-titer vector production in serum-free conditions while reducing the amount of input DNA required, resulting in a, highly efficient manufacturing option.

  • Liver-directed but not muscle-directed AAV-antibody gene transfer limits humoral immune responses in rhesus monkeys
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-11-26
    Sebastian P. Fuchs, José M. Martinez-Navio, Eva G. Rakasz, Guangping Gao, Ronald C. Desrosiers

    A number of publications have described the use of adeno-associated virus (AAV) for the delivery of anti-HIV and anti-SIV monoclonal antibodies (mAbs) to rhesus monkeys. Anti-drug antibodies (ADAs) have been frequently observed and long-term AAV-mediated delivery has been inconsistent. Here we investigated different AAV vector strategies and delivery schemes to rhesus monkeys using the rhesus monkey mAb 4L6. We compared 4L6 IgG1 delivery using the AAV1 vs. the AAV8 serotype with a CMV promoter and the use of a muscle-specific vs. a liver-specific promoter. Long-term expression levels of 4L6 IgG1 following AAV8-mediated gene transfer were comparable to those following AAV1-mediated gene transfer. AAV1-mediated gene transfer using a muscle-specific promoter showed robust ADAs and transiently low 4L6 IgG1 levels that ultimately declined to below detectable levels. Intravenous AAV8-mediated gene transfer using a liver-specific promoter also resulted in low levels of delivered 4L6 IgG1, but those low levels were maintained in the absence of any detectable ADAs. Booster injections using AAV1-CMV allowed for increased 4L6 IgG1 serum levels in animals that were primed with AAV8 but not with AAV1. Our results suggest that liver-directed expression may help to limit ADAs and that re-administration of AAV of a different serotype can result in successful long-term delivery of an immunogenic antibody.

  • Cross-Packaging and Capsid Mosaic Formation in Multiplexed AAV Libraries
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-11-26
    Pauline F. Schmit, Simon Pacouret, Eric Zinn, Elizabeth Telford, Fotini Nicolaou, Frédéric Broucque, Eva Andres-Mateos, Ru Xiao, Magalie Penaud-Budloo, Mohammed Bouzelha, Nicolas Jaulin, Oumeya Adjali, Eduard Ayuso, Luk H. Vandenberghe

    Generation and screening of libraries of adeno-associated virus (AAV) variants has emerged as a powerful method for identifying novel capsids for gene therapy applications. For the majority of libraries, vast population diversity requires multiplexed production, in which a library of ITR-containing plasmid variants is transfected together into cells to generate the viral library. This process has the potential to be confounded by cross-packaging and mosaicism, in which particles are comprised of genomes and capsid monomers derived from different library members. Here, we investigate the prevalence of cross-packaging and mosaicism in simplified, minimal libraries using novel assays designed to assess capsid composition and packaging fidelity. We show that AAV library variants are prone to cross-packaging and capsid mosaic formation when produced at high plasmid levels, although to a lesser extent than in a recombinant context. We also provide experimental evidence that dilution of input library DNA significantly increases capsid monomer homogeneity and increases capsid:genome correlation in AAV libraries. Lastly, we determine that similar dilution methods yield higher-quality libraries when used for in vivo screens. Together, these findings quantitatively characterized the prevalence of crosspackaging and mosaicism in AAV libraries and established conditions that minimize related noise in subsequent screens.

  • Three-Phase Partitioning Combined with Density Gradient Ultracentrifugation as an Economic and Universal Process for Large Scale Purification of AAV Vectors
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-11-22
    Zhe Yu, Siyun Zhou, Ningguang Luo, Ching Yi Ho, Min Chen, Haifeng Chen

    Adeno-associated virus (AAV) vectors have been commonly purified through density gradient ultracentrifugation (DGUC) or column chromatography methods. Though DGUC method can efficiently separate the empty from the full virus particles, its application in large scale AAV purification is hindered due to its limitation in volume of each centrifuge tube. In another hand, column chromatography is serotype-dependent, expensive and complicated, which co-purifies both empty and full virus particles. We describe here an economic and universal process using three-phase partitioning (TPP) combined with DGUC to purify large quantities of AAV vectors. First, TPP is used to remove up to 90% of the cellular impurities in the cell lysate and at the same time condense the AAV vectors into ∼10% of its original lysate volume. Second, two rounds of DGUC are employed to separate the empty from the full virus particles and at the same time remove the remaining cellular impurities. This combinational process increases the capacity of ultracentrifugation by a factor of 5 to 10 folds depending on the yields of AAV serotypes. A variety of AAV serotypes such as AAV2, AAV5, AAV6, AAV9, and AAVDJ has been successfully purified with this process. Both in vitro and in vivo studies demonstrate that TPP has no detrimental impact on AAV infectivity. In a proof of concept, we performed several purification runs ranging from 3 to 25 liters of Sf9 culture volume. We were able to purify more than 3e+15vg AAV vectors from 3 liters of cell culture volume with just two SW28 centrifuge tubes in a Beckman Coulter ultracentrifuge. Our data indicate that this TPP-DGUC process is economic, universal, and can be used to purify large quantity of AAV vectors for clinical applications with just a few ultracentrifuges.

  • Prednisolone Does Not Regulate Factor VIII Expression in Mice Receiving AAV5-hFVIII-SQ: Valoctocogene Roxaparvovec
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-11-21
    Lening Zhang, Britta Handyside, Ryan Murphy, Choong-Ryoul Sihn, Lin Xie, Catherine Vitelli, Danielle Harmon, Sílvia Sisó, Su Liu, Sherry Bullens, Stuart Bunting, Sylvia Fong

    AAV5-hFVIII-SQ (valoctocogene roxaparvovec) is an adeno-associated virus (AAV)-mediated gene therapy vector containing a B-domain-deleted human factor VIII (hFVIII-SQ) transgene. In a phase 1/2 clinical study of AAV5-hFVIII-SQ for severe hemophilia A (FVIII <1 IU/dL), participants received prednisolone to mitigate potential immune-mediated reactions to the gene therapy and demonstrated concomitant elevations in plasma FVIII levels, following a single administration of AAV5-hFVIII-SQ. To assess whether prednisolone is capable of directly modulating transgene expression or levels of circulating hepatic enzymes, C57BL/6 mice were given intravenous vehicle, 2×1013 vg/kg AAV5-hFVIII-SQ, or 6×1013 vg/kg AAV5-hFVIII-SQ, followed by either daily oral prednisolone or water. Mice were euthanized four or 13-weeks after vector administration. Hepatic hFVIII-SQ DNA, RNA, protein (immunostaining); plasma hFVIII-SQ protein and FVIII activity; aspartate aminotransferase (AST); and alanine aminotransferase (ALT); were measured. Liver hFVIII-SQ DNA, RNA, and plasma hFVIII-SQ protein and activity increased in a dose-dependent manner, with or without prednisolone. In summary, chronic prednisolone treatment in mice treated with AAV5-hFVIII-SQ did not modulate levels of liver hFVIII-SQ DNA, RNA, or percentage and distribution of hFVIII-SQ positive hepatocytes; nor did it regulate levels of plasma hFVIII-SQ protein or activity, or affect levels of plasma AST or ALT.

  • Establishment of SLC15A1/PEPT1-knockout human induced pluripotent stem cell line for intestinal drug absorption studies
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-11-21
    Kanae Kawai, Ryosuke Negoro, Moe Ichikawa, Tomoki Yamashita, Sayaka Deguchi, Kazuo Harada, Kazumasa Hirata, Kazuo Takayama, Hiroyuki Mizuguchi

    Because many peptide and peptide-mimetic drugs are substrates of peptide transporter 1, it is important to evaluate the peptide transporter 1-mediated intestinal absorption of drug candidates in the early phase of drug development. Although intestinal cell lines treated with inhibitors of peptide transporter 1 are widely used to examine whether drug candidates are substrates for peptide transporter 1, these inhibitors are not sufficiently specific for peptide transporter 1. Here, to generate a more precise evaluation model, we established peptide transporter 1-knockout iPS cells by using CRISPR-Cas9 system and differentiated the cells into intestinal epithelial-like cells. The permeability value and uptake capacity of glycylsarcosine (substrate of peptide transporter 1) in peptide transporter 1- knockout intestinal epithelial-like cells were significantly lower than those in wild-type intestinal epithelial-like cells, suggesting that peptide transporter 1 was successfully depleted in the epithelial cells. Taken together, our model can be useful in the development of peptide and peptide-mimetic drugs.

  • rAAV2-retro enables extensive and high-efficient transduction of lower motor neurons following intramuscular injection
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-11-21
    Zhilong Chen, Guoqing Fan, Anan Li, Jing Yuan, Tonghui Xu

    The motor system controls muscle movement through lower motor neurons in the spinal cord and brainstem. Lower motor neurons are efferent neurons in the central nervous system (CNS) characterized by axonal projections that reach specific targets in the periphery. Lower motor neuron lesions result in the denervation and dysfunction of peripheral skeletal muscle. Great progress has been made to develop therapeutic strategies to transduction lower motor neurons with genes. However, the widespread distribution of lower motor neurons makes their specific, extensive and efficient transduction a challenge. Here, we demonstrated that, compared to the other tested rAAV serotypes, rAAV2-retro mediated the most efficient retrograde transduction of lower motor neurons in the spinal cord following intramuscular injection in neonatal mice. A single injection of rAAV2-retro in a single muscle enabled the efficient and extensive transduction of lower motor neurons in the spinal cord and brainstem rather than transducing only the lower motor neurons connected to the injected muscle. rAAV2-retro achieved the extensive transduction of lower motor neurons by the cerebrospinal fluid pathway. Our work suggests that gene delivery via the intramuscular injection of rAAV2-retro represents a promising tool in the development of gene therapy strategies for motor neuron diseases.

  • Inclusion of PF68 surfactant improves stability of rAAV titre when passed through a surgical device used in retinal gene therapy clinical trials
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-11-20
    Maria I. Patrício, Christopher I. Cox, Clare Blue, Alun R. Barnard, Cristina Martinez-Fernandez Dela Camara, Robert E. MacLaren

    Recent advances in recombinant adeno-associated virus (rAAV) gene therapy for choroideremia show gene replacement to be a promising approach. It is however well known that contact of vector solution with plastic materials in the surgical device may result in non-specific adsorption with resulting loss of physical tire and/or level of protein expression and activity. Here we assessed the biocompatibility and stability of rAAV2-REP1 (Rab Escort Protein-1) before and following passage through the injection device over a period of time to mimic the clinical scenario. Three identical devices were screened using two concentrations of vector: high (1E+12 DRP/mL) and low (1E+11 DRP/mL) to mimic high and low dose administrations of vector product. The low dose was prepared using either formulation buffer that contained 0.001% of a non-ionic surfactant (PF68), or balanced salt solution (BSS). We observed significant losses in the genomic titre of samples diluted with BSS for all time-points. The addition of 0.001% PF68 did not however affect AAV physical titre nor REP1 protein expression and biological activity. Hence we observed that neither the genomic titre nor the biological activity of a rAAV2-REP1-containing solution was affected following passage through the surgical device when PF68 was present as a surfactant and this was maintained over a period up to 10 hours.

  • Dosing and re-administration of intravenous lentiviral vector for in vivo gene therapy: Studies in rhesus monkeys and ADA-deficient mice
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-11-16
    Denise A. Carbonaro-Sarracino, Alice F. Tarantal, C. Chang I. Lee, Michael L. Kaufman, Stephen Wandro, Xiangyang Jin, Michele Martinez, Danielle N. Clark, Krista Chun, Colin Koziol, Cinnamon L. Hardee, Xiaoyan Wang, Donald B. Kohn

    ADA-deficient mice and healthy rhesus monkeys were studied to determine the impact of age at treatment, vector dosage, dosing schedule, repeat administration, biodistribution, and immunogenicity after systemic delivery of lentiviral vectors (LV). In Ada-/- mice, neonatal treatment resulted in broad vector marking across all tissues analyzed, whereas adult treatment resulted in marking restricted to the liver, spleen, and bone marrow. Intravenous administration to infant rhesus monkeys also resulted in dose-dependent marking in the liver, spleen, and bone marrow. Using an ELISA to monitor anti-vector antibody development, Ada-/- neonatal mice did not produce an antibody response, whereas Ada-/- adult mice produced a strong antibody response to vector administration. In mice and monkeys with repeat administration of LV, a strong anti-vector antibody response was shown in response to the second LV administration which resulted in LV inactivation. ADA-deficient mice administered three separate doses resulted in acute toxicity. PEGylation of the VSV-G enveloped LV showed a less robust anti-vector response but did not prevent the inactivation of the second LV administration. These studies identify important factors to consider related to age and timing of administration when implementing systemic delivery of LV as a potential therapeutic agent.

  • Adipose Mesenchymal Cells-Derived Extracellular Vesicles Alleviate DOCA-salt-Induced Hypertension by Promoting Cardio-Renal Protection
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-11-15
    Rafael Soares Lindoso, Jarlene Alécia Lopes, Renata Binato, Eliana Abdelhay, Christina Maeda Takiya, Kildare Rocha de Miranda, Lucienne Silva Lara, Antonella Viola, Benedetta Bussolati, Adalberto Vieyra, Federica Collino

    Hypertension is a long-term condition that can increase organ susceptibility to insults and lead to severe complications such as chronic kidney disease (CKD). Extracellular vesicles (EVs) are cell-derived membrane structures that participate in cell-cell communication by exporting encapsulated molecules to target cells, regulating physiological and pathological processes. We demonstrate that multiple administration of EVs from adipose-derived mesenchymal stromal cells (ASC-EVs) in deoxycorticosterone acetate (DOCA)-salt hypertensive model can protect renal tissue by maintaining its filtration capacity. Indeed, ASC-EVs downregulated the pro-inflammatory molecules monocyte chemoattracting protein-1 and plasminogen activating inhibitor-1, and reduced recruitment of macrophage in the kidneys. Moreover, ASC-EVs prevented cardiac tissue fibrosis and maintained blood pressure within normal levels, thus demonstrating its multiple favorable effects in different organs. By applying miRNA microarray profile of the kidney of DOCA-salt rats, we identified a selective miRNA signature associated with epithelial-mesenchymal transition (EMT). One of the key pathways found was the axis miR-200-TGF-β, that was significantly altered by EV administration, thereby affecting the EMT signaling and preventing renal inflammatory response and fibrosis development. Our results indicate that EVs can be a potent therapeutic tool for the treatment of hypertension-induced CKD in cardio-renal syndrome.

  • A flow-cytometry platform for intracellular detection of FVIII in blood cells: a new tool to assess gene therapy efficiency for Hemophilia-A
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-11-15
    Muhammad Elnaggar, Anjud Al-Mohannadi, Dhanya Kizhakayil, Christophe Michel Raynaud, Sharefa Al-Mannai, Giusy Gentilcore, Igor Pavlovski, Abbirami Sathappan, Nicholas Van Panhuys, Chiara Borsotti, Antonia Follenzi, Jean-Charles Grivel, Sara Deola
  • Immunology of Adenoviral Vectors in Cancer Therapy
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-11-13
    Amanda Rosewell Shaw, Masataka Suzuki

    Adenoviruses are a commonly utilized virus for gene therapy-platforms worldwide. Since adenovirus components are characterized as highly immunogenic, their immunogenicity inhibits the widespread use of adenoviral vectors to treat genetic disorders. However, stimulation of the immune response can be exploited for cancer immunotherapy platforms, thus adenoviral vectors are used for therapeutic gene transfer, vaccines, and oncolytic agents in the cancer gene therapy field. It is now accepted that the generation of anti-tumor immune responses induced by oncolytic adenovirus treatments is critical for their anti-tumor efficacy. As such, in cancer immunotherapy with adenoviral vectors, a balance must be struck between induction of anti-adenoviral and anti-tumor immune responses. The recent trend in adenoviral-based cancer gene therapy is the development of adenoviral vectors to enhance immune responses and redirect them towards tumors. This review focuses on anti-adenoviral immunity and how adenovirotherapies skew the immune response toward an anti-tumor response.

  • Low-dose busulfan reduces human CD34+ cell doses required for engraftment in c-kit mutant immunodeficient mice
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-11-11
    Alexis Leonard, Morgan Yapundich, Tina Nassehi, Jackson Gamer, Claire M. Drysdale, Juan J. Haro-Mora, Selami Demirci, Matthew M. Hsieh, Naoya Uchida, John F. Tisdale

    Humanized animal models are central to efforts aimed at improving hematopoietic stem cell (HSC) transplantation with or without genetic modification. Human cell engraftment is feasible in immunodeficient mice; however, high HSC doses and conditioning limit broad use of xenograft models. We assessed human CD45+ chimerism after transplanting varying doses of human CD34+ HSCs (2x105-2x106 cells/mouse) with or without Busulfan (BU) pretransplant conditioning in c-kit mutant mice that do not require conditioning (NBSGW). We then tested a range of BU (5-37.5mg/kg) using 2x105 human CD34+ cells. Glycophorin-A erythrocyte chimerism was assessed after murine macrophage depletion using clodronate liposomes. We demonstrated successful long-term engraftment of human CD34+ cells at all cell doses in this model, and equivalent engraftment using tenfold less CD34+ cells with the addition of BU conditioning. Low-dose BU (10mg/kg) was sufficient to allow human engraftment using 2x105 CD34+ cells, whereas higher doses (≥37.5mg/kg) were toxic. NBSGW mice support human erythropoiesis in the bone marrow; however, murine macrophage depletion provided only minimal and transient increases in peripheral blood human erythrocytes. Our xenograft model is therefore useful in HSC gene therapy and genome editing studies, especially for modeling in disorders such as sickle cell disease where access to HSCs is limited.

  • Pathogenesis of hepatic tumors following gene therapy in murine and canine models of glycogen storage disease
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-11-11
    Hye-Ri Kang, Monika Gjorgjieva, Stephanie N. Smith, Elizabeth D. Brooks, Zelin Chen, Shawn M. Burgess, Randy J. Chandler, Lauren R. Waskowicz, Kylie M. Grady, Songtao Li, Gilles Mithieux, Charles P. Venditti, Fabienne Rajas, Dwight D. Koeberl

    Glycogen storage disease type Ia (GSD Ia) is caused by mutations in the glucose-6-phosphatase (G6Pase) catalytic subunit gene (G6PC). GSD Ia complications include hepatocellular adenomas (HCA) with a risk for hepatocellular carcinoma (HCC) formation. Genome editing with adeno-associated virus (AAV) vectors containing a zinc-finger nuclease (ZFN) and a G6PC donor transgene was evaluated in adult mice with GSD Ia. Although mouse livers expressed G6Pase, HCA and HCC occurred following AAV vector administration. Interestingly, vector genomes were almost undetectable in the tumors, but remained relatively high in adjacent liver (p<0.01). G6Pase activity was decreased in tumors, in comparison with adjacent liver (p<0.01). Furthermore, AAV-G6Pase vector-treated dogs with GSD Ia developed HCC with lower G6Pase activity (p<0.01), in comparison with adjacent liver. AAV integration and tumor marker analysis in mice revealed that tumors arose from the underlying disorder, not from vector administration Similarly to human GSD Ia–related HCA and HCC, mouse and dog tumors did not express elevated α-fetoprotein. Taken together, these results suggest that AAV-mediated gene therapy not only corrects hepatic G6Pase deficiency, but also has potential to suppress HCA and HCC in the GSD Ia liver.

  • AAV-mediated gene augmentation therapy restores critical functions in mutant PRPF31+/- iPSC-derived RPE cells.
    Mol. Ther. Methods Clin. Dev. (IF 4.875) Pub Date : 2019-11-11
    Elizabeth M. Brydon, Revital Bronstein, Adriana Buskin, Majlinda Lako, Eric A. Pierce, Rosario Fernandez-Godino
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