Anna Stornaiuolo

Failure to correct murine muscular dystrophy.

Bone-marrow cells have the potential to differentiate into other cell types such as muscle fibres, and can be transplanted into acutely or chronically damaged muscle as a way of delivering normal dystrophin (the protein that is defective or missing in Duchennes muscular dystrophy) to the skeletal and heart muscle of mdx mice, an animal model for this disease. But the corrective potential of this approach has been hard to estimate against the high background of muscle fibres that spontaneously revert to synthesizing dystrophin, a feature of the original mdx mutation. Here we test the long-term efficacy of bone-marrow transplantation in a different mdx mutant which is free of this problem and find that it has no impact on murine muscular dystrophy.

IL4 gene delivery to the CNS recruits regulatory T cells and induces clinical recovery in mouse models of multiple sclerosis

Central nervous system (CNS) delivery of anti-inflammatory cytokines, such as interleukin 4 (IL4), holds promise as treatment for multiple sclerosis (MS). We have previously shown that short-term herpes simplex virus type 1-mediated IL4 gene therapy is able to inhibit experimental autoimmune encephalomyelitis (EAE), an animal model of MS, in mice and non-human primates. Here, we show that a single administration of an IL4-expressing helper-dependent adenoviral vector (HD-Ad) into the cerebrospinal fluid (CSF) circulation of immunocompetent mice allows persistent transduction of neuroepithelial cells and long-term (up to 5 months) CNS transgene expression without toxicity. Mice affected by chronic and relapsing EAE display clinical and neurophysiological recovery from the disease once injected with the IL4-expressing HD-Ad vector. The therapeutic effect is due to the ability of IL4 to increase, in inflamed CNS areas, chemokines (CCL1, CCL17 and CCL22) capable of recruiting regulatory T cells (CD4+CD69−CD25+Foxp3+) with suppressant functions. CSF delivery of HD-Ad vectors expressing anti-inflammatory molecules might represent a valuable therapeutic option for CNS inflammatory disorders.

RD2-MolPack-Chim3, a Packaging Cell Line for Stable Production of Lentiviral Vectors for Anti-HIV Gene Therapy

Over the last two decades, several attempts to generate packaging cells for lentiviral vectors (LV) have been made. Despite different technologies, no packaging clone is currently employed in clinical trials. We developed a new strategy for LV stable production based on the HEK-293T progenitor cells; the sequential insertion of the viral genes by integrating vectors; the constitutive expression of the viral components; and the RD114-TR envelope pseudotyping. We generated the intermediate clone PK-7 expressing constitutively gag/pol and rev genes and, by adding tat and rd114-tr genes, the stable packaging cell line RD2-MolPack, which can produce LV carrying any transfer vector (TV). Finally, we obtained the RD2-MolPack-Chim3 producer clone by transducing RD2-MolPack cells with the TV expressing the anti-HIV transgene Chim3. Remarkably, RD114-TR pseudovirions have much higher potency when produced by stable compared with transient technology. Most importantly, comparable transduction efficiency in hematopoietic stem cells (HSC) is obtained with 2-logs less physical particles respect to VSV-G pseudovirions produced by transient transfection. Altogether, RD2-MolPack technology should be considered a valid option for large-scale production of LV to be used in gene therapy protocols employing HSC, resulting in the possibility of downsizing the manufacturing scale by about 10-fold in respect to transient technology.

Absence of an intrathecal immune reaction to a helper-dependent adenoviral vector delivered into the cerebrospinal fluid of non-human primates

Inflammation and immune reaction, or pre-existing immunity towards commonly used viral vectors for gene therapy severely impair long-term gene expression in the central nervous system (CNS), impeding the possibility to repeat the therapeutic intervention. Here, we show that injection of a helper-dependent adenoviral (HD-Ad) vector by lumbar puncture into the cerebrospinal fluid (CSF) of non-human primates allows long-term (three months) infection of neuroepithelial cells, also in monkeys bearing a pre-existing anti-adenoviral immunity. Intrathecal injection of the HD-Ad vector was not associated with any sign of systemic or local toxicity, nor by signs of a CNS-specific immune reaction towards the HD-Ad vector. Injection of HD-Ad vectors into the CSF circulation may thus represent a valuable approach for CNS gene therapy allowing for long-term expression and re-administration.

RD-MolPack technology for the constitutive production of self-inactivating lentiviral vectors pseudotyped with the nontoxic RD114-TR envelope

To date, gene therapy with transiently derived lentivectors has been very successful to cure rare infant genetic diseases. However, transient manufacturing is unfeasible to treat adult malignancies because large vector lots are required. By contrast, stable manufacturing is the best option for high-incidence diseases since it reduces the production cost, which is the major current limitation to scale up the transient methods. We have previously developed the proprietary RD2-MolPack technology for the stable production of second-generation lentivectors, based on the RD114-TR envelope. Of note, opposite to vesicular stomatitis virus glycoprotein (VSV-G) envelope, RD114-TR does not need inducible expression thanks to lack of toxicity. Here, we present the construction of RD2- and RD3-MolPack cells for the production of self-inactivating lentivectors expressing green fluorescent protein (GFP) as a proof-of-concept of the feasibility and safety of this technology before its later therapeutic exploitation. We report that human T lymphocytes transduced with self-inactivating lentivectors derived from RD3-MolPack cells or with self-inactivating VSV-G pseudotyped lentivectors derived from transient transfection show identical T-cell memory differentiation phenotype and comparable transduction efficiency in all T-cell subsets. RD-MolPack technology represents, therefore, a straightforward tool to simplify and standardize lentivector manufacturing to engineer T-cells for frontline immunotherapy applications.

Codon Optimization Leads to Functional Impairment of RD114-TR Envelope Glycoprotein

Lentiviral vectors (LVs) are a highly valuable tool for gene transfer currently exploited in basic, applied, and clinical studies. Their optimization is therefore very important for the field of vectorology and gene therapy. A key molecule for LV function is the envelope because it guides cell entry. The most commonly used in transiently produced LVs is the vesicular stomatitis virus glycoprotein (VSV-G) envelope, whose continuous expression is, however, toxic for stable LV producer cells. In contrast, the feline endogenous retroviral RD114-TR envelope is suitable for stable LV manufacturing, being well tolerated by producer cells under constitutive expression. We have previously reported successful, transient and stable production of LVs pseudotyped with RD114-TR for good transduction of T lymphocytes and CD34+ cells. To further improve RD114-TR-pseudotyped LV cell entry by increasing envelope expression, we codon-optimized the RD114-TR open reading frame (ORF). Here we show that, despite the RD114-TRco precursor being produced at a higher level than the wild-type counterpart, it is unexpectedly not duly glycosylated, exported to the cytosol, and processed. Correct cleavage of the precursor in the functional surface and transmembrane subunits is prevented in vivo, and, consequently, the unprocessed precursor is incorporated into LVs, making them inactive.

Extracellular NGFR Spacers Allow Efficient Tracking and Enrichment of Fully Functional CAR-T Cells Co-Expressing a Suicide Gene

Chimeric antigen receptor (CAR)-T cell immunotherapy is at the forefront of innovative cancer therapeutics. However, lack of standardization of cellular products within the same clinical trial and lack of harmonization between different trials have hindered the clear identification of efficacy and safety determinants that should be unveiled in order to advance the field. With the aim of facilitating the isolation and in vivo tracking of CAR-T cells, we here propose the inclusion within the CAR molecule of a novel extracellular spacer based on the low-affinity nerve-growth-factor receptor (NGFR). We screened four different spacer designs using as target antigen the CD44 isoform variant 6 (CD44v6). We successfully generated NGFR-spaced CD44v6 CAR-T cells that could be efficiently enriched with clinical-grade immuno-magnetic beads without negative consequences on subsequent expansion, immuno-phenotype, in vitro antitumor reactivity, and conditional ablation when co-expressing a suicide gene. Most importantly, these cells could be tracked with anti-NGFR monoclonal antibodies in NSG mice, where they expanded, persisted, and exerted potent antitumor effects against both high leukemia and myeloma burdens. Similar results were obtained with NGFR-enriched CAR-T cells specific for CD19 or CEA, suggesting the universality of this strategy. In conclusion, we have demonstrated that the incorporation of the NGFR marker gene within the CAR sequence allows for a single molecule to simultaneously work as a therapeutic and selection/tracking gene. Looking ahead, NGFR spacer enrichment might allow good manufacturing procedures-manufacturing of standardized CAR-T cell products with high therapeutic potential, which could be harmonized in different clinical trials and used in combination with a suicide gene for future application in the allogeneic setting.

398. Transduction of Human Hematopoietic Stem Cells and T-Lymphocytes with RD114-TR-Pseudotyped Lentiviral Vectors

Lentiviral vectors transduce at high efficiency human hematopoietic stem/progenitor cells (HSCs) without compromising their self-renewing and repopulation capacity. Last-generation helper constructs and self-inactivating (SIN) vectors have minimized the chances of generating recombinant, replication-competent HIV derivatives during packaging, and drastically reduced the potential risks of insertional activation of oncogenes. To allow the clinical use of lentiviral vectors, development of packaging cell lines and standardized procedures for vector manufacturing, downstream processing and quality/safety testing appears necessary. However, generation of stable packaging cell lines has been hampered so far by the high toxicity of VSV-G and of some of the HIV helper proteins used in the packaging steps. More recently, lentiviral vectors have been pseudotyped with alternative, non-toxic envelope proteins such as derivatives of the RD114 feline leukemia virus envelope glycoprotein, allowing the generation of a prototype, stable packaging cell line (Ikeda et al., Nature Biotech 21:569, 2003). The receptor for RD114 is abundant on human hematopoietic cells, allowing efficient transduction by RD114-pseudotyped lentiviral vectors. We report a comparison between lentiviral vector preparations pseudotyped with VSV-G and the RD114-TR fusion glycoprotein in transducing human cord blood-derived CD34+ HSCs and peripheral blood lymphocytes (PBLs). RD114-TR-pseudotyped vectors transduce both cell types at much lower m.o.i than VSV-G-pseudotyped vectors (5–10 vs. 50–200), resulting in lower toxicity, higher persistence and less vector copy number per genome. In addition, RD114-TR-pseudotyped vectors transduce at higher efficiency human clonogenic progenitors (BFU-E, CFU-GM, CFU-GEMM), as assayed in semi-solid cultures in vitro. Transduction efficiency and average vector copy number in human repopulating HSCs and their progeny was determined after xeno-transplantation in NOD-SCID mice. Our results indicate that pseudotyping with RD114-TR is a promising alternative to VSV-G for developing clinical-grade lentiviral vector preparations aimed at genetic modification of human HSCs.