Mutation-independent treatment of autosomal dominant Retinitis Pigmentosa (adRP)

Viral-mediated gene therapy holds great promise for the treatment of severe inherited retinal diseases, such as Retintitis Pigmentosa (RP), which is caused by mutations in genes preferentially expressed in photoreceptor cells. The availability of vectors derived from the small adeno-associated virus (AAV) which efficiently and stably transduce the retina of animal models after intraocular administration strongly support the possibility to develop novel strategies for the treatment of such severe retinal degenerations otherwise incurable thus far. The main goals of my PhD project were: - generate artificial transcription repressors (ZFPs) targeted to the human rhodopsin promoter to silence at the transcriptional level the rhodopsin gene; - assess the efficacy of the treatment and the impact on the disease progression in the RP mouse model. Retinitis pigmentosa is by far the most studied inherited retinal disease. It is clinically and genetically heterogeneous recognizing autosomal recessive (arRP), autosomal dominant (adRP), X-linked, and digenic patterns of inheritance. More than 30 diseases genes have been identified so far and 12 of these have been associated with (adRP), representing between 15% and 35% of all cases. Despite recent success of the gene-based complementation approach for genetic recessive traits, the development of therapeutic strategies for gain-of-function mutations poses great challenges. General therapeutic principles to correct these genetic defects mostly rely on post-transcriptional gene regulation (RNA silencing). Engineered zinc finger protein (ZFP)-based-repression of transcription may represent a novel and alternative mutation independent therapeutic approach for treating gain-of-function mutations, but proof-of-concept of this use is still lacking. In my PhD project we used a novel strategy to treat adRP based on zinc-finger-based artificial transcription factors (ZF-ATFs). These molecules can be engineered to silence genes carrying gain-of-function mutations that cause toxic effects into the cell where they are expressed. We generated ten artificial transcriptional repressors targeted to the human Rhodopsin which is the gene most commonly associated with adRP (20–30% of cases) with more than 150 mutations identified throughout its sequence, representing the most commonly mutated gene in RP. We characterized in vitro the ability of artificial transcriptional repressors to bind specifically the human rhodopsin promoter in order to exert a specific transcriptional control and we selected two out of ten functional zinc-finger-based repressors of rhodopsin. One of this was selected as the most efficient and was enclosed in an AAV2/8 for in vivo experiments. We demonstrated that the selected artificial zinc-finger-based repressors (ZFRs) resulted in a robust transcriptional repression of hRHO impacting disease progression in a mouse model of adRP over-expressing the P347S mutation. The data obtained support the use of ZFP-mediated silencing as a potentially relevant therapeutic strategy to treat gain of function mutations

125. Engineered Nucleases-Mediated In Situ Correction of a Genetic Defect By Homologous Recombination Into the Native Locus

Engineered nucleases specific for genomic targets are extensively used to generate DSBs that increase the rate and efficiency of homologous recombination (HR). We seek to determine the efficacy of nucleases in a clinical relevant genetic defect.The genetic defect we are addressing as model to test the nucleases-mediated genome editing technology is the junctional epidermolysis bullosa (JEB), a family of severe skin adhesion disorders due to autosomal recessive mutations in the LAMB3 gene coding for the laminin-332 heterotrimer, a key component of the dermal-epidermaljunction. Recently, we provided proof of principle that ZFN-mediated, AAVS1-targeted GFP addition can be achieved in human keratinocytes and in long-term repopulating epithelial stem cells in a validated preclinical model of xenotransplantation of human skin equivalents on immunodeficient mice.This project aims at the demonstration of a successful in situ correction of the LAMB3 gene in primary keratinocytes from Herlitz JEB patients. Recently TALEN-based gene correction for dystrophic EB has been reported. Similarly, we have developed a genome editing approach for JEB. In particular we have designed TALENs specific for the second intron of LAMB3 gene and a HR cassette including a splicible LAMB3 cDNA (from exon 3 to the end of the gene). In particular immortalized JEB keratinocytes were transfected with TALEN mRNAs and infected with an IDLV vector carrying the HR cassette. The in situ gene correction has been evaluated by site-specific PCR and knock-in expression of the corrected LAMB3 gene on bulk population. We then assessed targeting efficiency and specificity by extensive molecular analyses of single-cell clones isolated by limiting dilution from the TALENs/IDLV-treated immortalized JEB population. We isolated 256 clones and expanded 69 of them. Sixteen out of 69 clones showed an in vitro adhesion advantage, hosted the HR cassette correctly integrated into the predetermined locus, expressed the corrected LAMB3 gene and produced the laminin-332 protein. In parallel, CRISPR-Cas9 nuclease has been designed on the same locus to compare the transduction efficiency and cleavage activity and to translate the knock-in targeting platform to primary JEB keratinocytes

TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity

Designer nucleases have been successfully employed to modify the genomes of various model organisms and human cell types. While the specificity of zinc-finger nucleases (ZFNs) and RNA-guided endonucleases has been assessed to some extent, little data are available for transcription activator-like effector-based nucleases (TALENs). Here, we have engineered TALEN pairs targeting three human loci (CCR5, AAVS1 and IL2RG) and performed a detailed analysis of their activity, toxicity and specificity. The TALENs showed comparable activity to benchmark ZFNs, with allelic gene disruption frequencies of 15-30% in human cells. Notably, TALEN expression was overall marked by a low cytotoxicity and the absence of cell cycle aberrations. Bioinformatics-based analysis of designer nuclease specificity confirmed partly substantial off-target activity of ZFNs targeting CCR5 and AAVS1 at six known and five novel sites, respectively. In contrast, only marginal off-target cleavage activity was detected at four out of 49 predicted off-target sites for CCR5- and AAVS1-specific TALENs. The rational design of a CCR5-specific TALEN pair decreased off-target activity at the closely related CCR2 locus considerably, consistent with fewer genomic rearrangements between the two loci. In conclusion, our results link nuclease-associated toxicity to off-target cleavage activity and corroborate TALENs as a highly specific platform for future clinical translation

Genome Editing With TALEN, CRISPR-Cas9 and CRISPR-Cas12a in Combination With AAV6 Homology Donor Restores T Cell Function for XLP

X-linked lymphoproliferative disease is a rare inherited immune disorder, caused by mutations or deletions in the SH2D1A gene that encodes an intracellular adapter protein SAP (Slam-associated protein). SAP is essential for mediating several key immune processes and the immune system - T cells in particular - are dysregulated in its absence. Patients present with a spectrum of clinical manifestations, including haemophagocytic lymphohistiocytosis (HLH), dysgammaglobulinemia, lymphoma and autoimmunity. Treatment options are limited, and patients rarely survive to adulthood without an allogeneic haematopoietic stem cell transplant (HSCT). However, this procedure can have poor outcomes in the mismatched donor setting or in the presence of active HLH, leaving an unmet clinical need. Autologous haematopoeitic stem cell or T cell therapy may offer alternative treatment options, removing the need to find a suitable donor for HSCT and any risk of alloreactivity. SAP has a tightly controlled expression profile that a conventional lentiviral gene delivery platform may not be able to fully replicate. A gene editing approach could preserve more of the endogenous regulatory elements that govern SAP expression, potentially providing a more optimum therapy. Here, we assessed the ability of TALEN, CRISPR-Cas9 and CRISPR-Cas12a nucleases to drive targeted insertion of SAP cDNA at the first exon of the SH2D1A locus using an adeno-associated virus serotype 6 (AAV6)-based vector containing the donor template. All nuclease platforms were capable of high efficiency gene editing, which was optimised using a serum-free AAV6 transduction protocol. We show that T cells from XLP patients corrected by gene editing tools have restored physiological levels of SAP gene expression and restore SAP-dependent immune functions, indicating a new therapeutic opportunity for XLP patients

Zinc-finger-based transcriptional repression of rhodopsin in a model of dominant retinitis pigmentosa

Despite the recent success of gene-based complementation approaches for genetic recessive traits, the development of therapeutic strategies for gain-of-function mutations poses great challenges. General therapeutic principles to correct these genetic defects mostly rely on post-transcriptional gene regulation (RNA silencing). Engineered zinc-finger (ZF) protein-based repression of transcription may represent a novel approach for treating gain-of-function mutations, although proof-of-concept of this use is still lacking. Here, we generated a series of transcriptional repressors to silence human rhodopsin (hRHO), the gene most abundantly expressed in retinal photoreceptors. The strategy was designed to suppress both the mutated and the wild-type hRHO allele in a mutational-independent fashion, to overcome mutational heterogeneity of autosomal dominant retinitis pigmentosa due to hRHO mutations. Here we demonstrate that ZF proteins promote a robust transcriptional repression of hRHO in a transgenic mouse model of autosomal dominant retinitis pigmentosa. Furthermore, we show that specifically decreasing the mutated human RHO transcript in conjunction with unaltered expression of the endogenous murine Rho gene results in amelioration of disease progression, as demonstrated by significant improvements in retinal morphology and function. This zinc-finger-based mutation-independent approach paves the way towards a ‘repression–replacement’ strategy, which is expected to facilitate widespread applications in the development of novel therapeutics for a variety of disorders that are due to gain-of-function mutations

Engineered zinc finger nickases induce homology-directed repair with reduced mutagenic effects

Engineered zinc finger nucleases (ZFNs) induce DNA double-strand breaks at specific recognition sequences and can promote efficient introduction of desired insertions, deletions or substitutions at or near the cut site via homology-directed repair (HDR) with a double- and/or single-stranded donor DNA template. However, mutagenic events caused by error-prone non-homologous end-joining (NHEJ)-mediated repair are introduced with equal or higher frequency at the nuclease cleavage site. Furthermore, unintended mutations can also result from NHEJ-mediated repair of off-target nuclease cleavage sites. Here, we describe a simple and general method for converting engineered ZFNs into zinc finger nickases (ZFNickases) by inactivating the catalytic activity of one monomer in a ZFN dimer. ZFNickases show robust strand-specific nicking activity in vitro. In addition, we demonstrate that ZFNickases can stimulate HDR at their nicking site in human cells, albeit at a lower frequency than by the ZFNs from which they were derived. Finally, we find that ZFNickases appear to induce greatly reduced levels of mutagenic NHEJ at their target nicking site. ZFNickases thus provide a promising means for inducing HDR-mediated gene modifications while reducing unwanted mutagenesis caused by error-prone NHEJ

Rescue of DNA-PK Signaling and T-Cell Differentiation by Targeted Genome Editing in a prkdc Deficient iPSC Disease Model

In vitro disease modeling based on induced pluripotent stem cells (iPSCs) provides a powerful system to study cellular pathophysiology, especially in combination with targeted genome editing and protocols to differentiate iPSCs into affected cell types. In this study, we established zinc-finger nuclease-mediated genome editing in primary fibroblasts and iPSCs generated from a mouse model for radiosensitive severe combined immunodeficiency (RS-SCID), a rare disorder characterized by cellular sensitivity to radiation and the absence of lymphocytes due to impaired DNA-dependent protein kinase (DNA-PK) activity. Our results demonstrate that gene editing in RS-SCID fibroblasts rescued DNA-PK dependent signaling to overcome radiosensitivity. Furthermore, in vitro T-cell differentiation from iPSCs was employed to model the stage-specific T-cell maturation block induced by the disease causing mutation. Genetic correction of the RS-SCID iPSCs restored T-lymphocyte maturation, polyclonal V(D)J recombination of the T-cell receptor followed by successful beta-selection. In conclusion, we provide proof that iPSC-based in vitro T-cell differentiation is a valuable paradigm for SCID disease modeling, which can be utilized to investigate disorders of T-cell development and to validate gene therapy strategies for T-cell deficiencies. Moreover, this study emphasizes the significance of designer nucleases as a tool for generating isogenic disease models and their future role in producing autologous, genetically corrected transplants for various clinical applications

A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity

Sequence-specific nucleases represent valuable tools for precision genome engineering. Traditionally, zinc-finger nucleases (ZFNs) and meganucleases have been used to specifically edit complex genomes. Recently, the DNA binding domains of transcription activator-like effectors (TALEs) from the bacterial pathogen Xanthomonas have been harnessed to direct nuclease domains to desired genomic loci. In this study, we tested a panel of truncation variants based on the TALE protein AvrBs4 to identify TALE nucleases (TALENs) with high DNA cleavage activity. The most favorable parameters for efficient DNA cleavage were determined in vitro and in cellular reporter assays. TALENs were designed to disrupt an EGFP marker gene and the human loci CCR5 and IL2RG. Gene editing was achieved in up to 45% of transfected cells. A side-by-side comparison with ZFNs showed similar gene disruption activities by TALENs but significantly reduced nuclease-associated cytotoxicities. Moreover, the CCR5-specific TALEN revealed only minimal off-target activity at the CCR2 locus as compared to the corresponding ZFN, suggesting that the TALEN platform enables the design of nucleases with single-nucleotide specificity. The combination of high nuclease activity with reduced cytotoxicity and the simple design process marks TALENs as a key technology platform for targeted modifications of complex genomes

Precise Epigenome Editing on the Stage: A Novel Approach to Modulate Gene Expression

In the last decades, a better understanding of human pathologies has revealed that genetic alterations as well as epigenetic aberrations can be drivers of a disease or exacerbate its manifestation. The availability of customizable platforms that allow precise genomic targeting has opened the possibility to cure genetic disorders by tackling directly the origin of the disease. Indeed, tethering of different effectors to a DNA-binding moiety grants precise alterations of the genome, transcriptome, or epigenome with the aim of normalizing disease-causing aberrations. The use of designer nucleases for therapeutic genome editing is currently approaching the clinics, and safety concerns arise with respect to off-target effects. Epigenome editing might be a valuable alternative, as it does not rely on DNA double-strand breaks, one of the most deleterious form of DNA damage, to exert its function. We have recently described designer epigenome modifier (DEM), a novel platform for achieving precise epigenome editing in clinically relevant primary human cells. We discuss the efficiency of DEM and highlight their remarkable safety profile, which certainly makes this platform a valuable candidate for future clinical translation