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There are several diseases whereby the goal of gene therapy is to silence rather than replace a gene function. Paradigmatic examples are diseases caused by a dominant negative mutation or those in which silencing of a host gene confers resistance to a pathogen or compensates the function of the missing gene. Yet, gene silencing can be used to enhance efficacy of cell therapy and for biotechnological applications. Until now, two technologies have been used to silence gene expression, namely RNA interference with short harping RNAs (shRNA) and gene disruption with Artificial Nucleases (ANs). Although some promising pre-clinical and clinical data have been already obtained, the low efficiency of knock-down with shRNA and of biallelic disruption with ANs may limit efficacy of these treatments, especially when residual gene activity can exert a biological function. To overcome this issue, we have developed a novel modality of gene silencing that exploits endogenous epigenetic mechanisms to convey robust and heritable states of repression at the desired target gene. We have generated Artificial Transcriptional Repressors (ATRs), chimeric proteins containing a custom-made DNA binding domain fused to the effector domain of a chromatinmodifying enzyme involved in silencing of Endogenous RetroViruses (ERVs). By performing iterative rounds of selection in human cell lines and primary cells engineered to report for synergistic activity of candidate effector domains, we identified a combination of 3 domains that, when transiently co-assembled on the promoter of the reporter cassette, fully abrogated transgene expression in up to 90% of treated cells. Importantly, silencing was maintained for more than 250 days in cultured cell lines, was resistant to in vitro differentiation or metabolic activation of primary cells, and was confined to the reporter cassette. Silencing was associated with high levels of de novo DNA methylation at the targeted locus and was dependent on this epigenetic mark for its propagation. Finally, transient transfection of 3 ATRs targeted to the promoter region of the Beta-2-microglobulin (B2M) gene resulted in the loss of surface expression of B2M and, consequently, of the MHC-I molecules in up to 80% of treated cells. This phenotype was associated with a switch in the epigenetic and transcriptional state of the constitutively active B2M gene, which became highly decorated with DNA methylation and deprived of RNA PolII and of its transcript. Of note, silencing was resistant to IFN-γ treatment, a potent B2M inducer. Overall, these data provide the first demonstration of efficient and stable silencing of an endogenous gene upon transient delivery of ATRs. This result was made possible by repurposing the machinery involved in silencing of ERVs, which instructs self-sustaining repressive epigenetic states on the gene of interest. While silencing of B2M might be used to generate universally transplantable allogeneic cells, our hit-and-run strategy provides a powerful new alternative to conventional gene silencing for the treatment of several diseases. (LN & AL co-authorship)

A novel strategy of targeted gene correction of the interleukin-2 receptor common gamma chain (IL2RG) gene for the treatment of X-linked Severe Combined Immunodeficiency (SCID-X1) is achieved by the combination of a pair of IL2RG-specific Zinc Finger Nucleases (ZFN) and the correct-gene template DNA delivered by integration-defective lentiviral vector (IDLV).The transient expression of the ZFN pair targeting the disease-causing gene is obtained by the electroporation of the two corresponding mRNAs, produced by in vitro transcription starting from plasmid DNA template. A major limitation of the mRNA transcribed in vitro is the presence of residual contaminants such as short RNAs and double stranded (ds)RNAs that may affect the function and spectrophotometric quantification of the product hampering therefore the delivery of high quality and precise amount of mRNA to target cells. Moreover, dsRNA contaminants represent a possible risk in terms of immunogenicity of the product, leading to activation of unwanted innate immune response with consequent reduction/abrogation of mRNA translation as well as potential alteration of the properties of the transfected cells. To improve nuclease expression while decreasing cellular innate response to mRNA transfection we combined different strategies: (i) inclusion of UTRs and polyA tails in the DNA template used for mRNA production; (ii) use of modified nucleotides during mRNA production and (iii) purification of the mRNAs by dHPLC with a reverse phase column made of non-porous matrix consisting of polystyrene-divinylbenzene copolymer beads alkylated with C-18 chains (Transgenomic, LTD.). In particular, the purification of in vitro transcribed mRNAs by means of dHPLC has been shown to strongly improve the translation of mRNA and significantly reduce the contaminant presence thus preventing innate immunity and eventually increasing modified cells persistence in vivo. We have developed feasible and reproducible, small and large scale mRNA production and downstream purification processes of the ZFN pairs obtaining accurate RNA quantification and reduced risk of immunogenicity. The full process achieved a 60% yield, loading with a 500µg RNA for each run with a single clean chromatographic peak. Furthermore, the level of residual organic solvent (i.e. Acetonitrile) used in the purification process is compatible with that applicable into clinic. The highly translatable non-immunogenic dHPLC-purified mRNA can be delivered without toxicity and represents a powerful and safe tool for the application of gene therapy protocols.

Lentiviral vectors (LVs) represent efficient and versatile vehicles for gene therapy. Current manufacturing of clinical-grade LVs mostly relies on transient transfection of plasmids expressing the multiple vector components. This method is labor and cost intensive and becomes challenging when facing the need of scale-up and standardization. The development of stable LV producer cell lines will greatly facilitate overcoming these hurdles. We have generated an inducible LV packaging cell line, carrying the genes encoding for third-generation vector components stably integrated in the genome under the control of tetracycline-regulated promoters. These LV packaging cells are stable in culture even after single-cell cloning and can be scaled up to large volumes. In order to minimize the immunogenicity of LVs for in vivo administration, we set out to remove the highly polymorphic class-I major histocompatibility complexes (MHC-I) expressed on LV packaging cells and incorporated in the LV envelope. We performed genetic disruption of the β-2 microglobulin (B2M) gene, a required component for the assembly and trafficking of all MHC-I to the plasma membrane in LV producer cells, exploiting the RNA-guided Cas9 nuclease. The resulting B2M-negative cells were devoid of surface-exposed MHC-I and produced MHC-free LVs. These LVs retain their infectivity on all tested cells in vitro and efficiently transduced the mouse liver upon intravenous administration. Strikingly, the MHC-free LVs showed significantly reduced immunogenicity in a T-cell activation assay performed on human primary T cells co-cultured with syngeneic monocytes exposed to LV, from several (n=7) healthy donors. To reproducibly generate LV-producer cell lines from these cells, we insert the LV genome of interest in the AAVS1 locus, chosen for robust expression, exploiting engineered nucleases and homology-directed repair. By this strategy, we have obtained several independent producer cell lines for LVs that express marker or therapeutic genes and are devoid of plasmid DNA contamination. LVs produced by these cells reproducibly show titer and infectivity within the lower bound range of standard optimized transient transfection, and effectively transduce relevant target cells, such as hematopoietic stem/progenitor cells and T cells ex vivo and the mouse liver in vivo. Overall, we provide evidence that rationally designed targeted genome engineering can be used to improve the yield, quality, safety and sustainability of LV production for clinical use.

Lentiviral vectors (LVs) represent efficient and versatile vehicles for gene therapy. The manufacturing of clinical-grade LVs relies on transient transfection of vector components. This method is labor and cost intensive and becomes challenging when facing the need of scale-up and standardization. The development of stable LV producer cell lines will greatly facilitate overcoming these hurdles. We have generated an inducible LV packaging cell line, carrying the genes encoding for third-generation vector components stably integrated in the genome under the control of tetracycline-regulated promoters. In order to minimize the immunogenicity of LVs for in vivo administration, we set out to remove the highly polymorphic and antigenic class-I major histocompatibility complex (MHC-I) expressed on LV packaging cells and subsequently incorporated on the LV envelope. We performed genetic disruption of the β-2 microglobulin (B2M) gene, a required component for the assembly and trafficking of the MHC-I to the plasma membrane in LV producer cells, exploiting the RNA-guided Cas9 nuclease. We generated B2M-negative cells devoid of surface-exposed MHC-I, which retain the ability to produce LVs. In order to insert the LV genome of interest in the packaging cell line, we performed site-specific integration in predetermined loci of the genome of these cells, chosen for robust expression, exploiting artificial nucleases and homology-directed repair. In several independent iterations of this process, we generated producer cell lines both for LV expressing marker genes and a therapeutic gene, i.e. coagulation factor IX (FIX), the gene mutated in hemophilia B. We show that these LV producer cells are stable in culture and can produce several liters of LV-containing conditioned medium. These LVs have comparable or only slightly lower infectious titer and specific infectivity than LVs produced by state-of-the-art transient transfection process and can transduce therapeutically relevant target cells, such as hematopoietic stem/progenitor cells and T lymphocytes to high efficiency. Moreover, we intravenously administered FIX-expressing LVs produced by the cell line to hemophilia B mice and established therapeutic levels of circulating FIX. These data indicate that site-specific integration is an efficient, rapid and reproducible method to generate LV producer cells, starting from a universal stable inducible LV packaging cell line. Overall, we provide evidence that rationally designed targeted genome engineering can be used to improve the quality, safety and sustainability of LV production for clinical use.