CRISPR/Cas9 technology offers novel approaches for the development of new therapies for many unmet clinical needs, including a significant number of inherited monogenic diseases. However, in vivo correction of disease-causing genes is still inefficient, especially for those diseases without selective advantage for corrected cells. We reasoned that substrate reduction therapies (SRT) targeting non-essential enzymes could provide an attractive alternative. Here we evaluate the therapeutic efficacy of an in vivo CRISPR/Cas9-mediated SRT to treat primary hyperoxaluria type I (PH1), a rare inborn dysfunction in glyoxylate metabolism that results in excessive hepatic oxalate production causing end-stage renal disease. A single systemic administration of an AAV8-CRISPR/Cas9 vector targeting glycolate oxidase, prevents oxalate overproduction and kidney damage, with no signs of toxicity in Agxt1−/− mice. Our results reveal that CRISPR/Cas9-mediated SRT represents a promising therapeutic option for PH1 that can be potentially applied to other metabolic diseases caused by the accumulation of toxic metabolites.

The development of advanced gene and cell therapies for the treatment of genetic diseases requires reliable animal and cellular models to test their efficacy. Moreover, the availability of the target human primary cells of these therapies is reduced in many diseases. The development of endonucleases that can cut into specific sites of the cell genome, as well as the repair of the generated break by non-homologous end-joining, results in a variety of outcomes, insertions, deletions, and inversions that can induce the disruption of any specific gene. Among the many methods that have been developed for gene editing, CRISPR-Cas9 technology has become one of the most widely used endonuclease tools due to its easy design and its low cost. It has also been reported that the use of two guides, instead of just the one required, reduces the outcomes of non-homologous end joining mainly to the precise genomic sequences between the cutting sites of the guides used. We have explored this strategy to generate useful cellular and animal models. Different distances between the two guides have been tested (from 8 to 500 bp apart), and using the optimal range of 30–60 bp we have obtained a human primary cellular model of a genetic disease, pyruvate kinase deficiency, where the availability of the target cells is limited. We have also generated an in vivo model of glycolate oxidase (GO) deficiency, which is an enzyme involved in the glyoxylate metabolism following the same strategy. We demonstrate that the use of two-guide CRISPR-Cas9-induced non-homologous end joining is a feasible and useful tool for disease modeling, and it is most relevant to those diseases in which it is difficult to get the cells that will be genetically manipulated.

Plasmodium multigene families are thought to play important roles in the pathogenesis of malaria. Plasmodium interspersed repeat (pir) genes comprise the largest multigene family in many Plasmodium species. However, their expression pattern and localisation remain to be elucidated. Understanding protein subcellular localisation is fundamental to reveal the functional importance and cell-cell interactions of the PIR proteins. Here, we use the rodent malaria parasite, Plasmodium chabaudi chabaudi, as a model to investigate the localisation pattern of this gene family. We found that most PIR proteins are co-expressed in clusters during acute and chronic infection; members of the S7 clade are predominantly expressed during the acute-phase, whereas members of the L1 clade dominate the chronic-phase of infection. Using peptide antisera specific for S7 or L1 PIRS, we show that these PIRs have different localisations within the infected red blood cells. S7 PIRs are exported into the infected red blood cell cytoplasm where they are co-localised with parasite-induced host cell modifications termed Maurer’s clefts, whereas L1 PIRs are localised on or close to the parasitophorous vacuolar membrane. This localisation pattern changes following mosquito transmission and during progression from acute- to chronic-phase of infection. The presence of PIRs in Maurer’s clefts, as seen for Plasmodium falciparum RIFIN and STEVOR proteins, might suggest trafficking of the PIRs on the surface of the infected erythrocytes. However, neither S7 nor L1 PIR proteins detected by the peptide antisera are localised on the surface of infected red blood cells, suggesting that they are unlikely to be targets of surface variant-specific antibodies or to be directly involved in adhesion of infected red blood cells to host cells, as described for Plasmodium falciparum VAR proteins. The differences in subcellular localisation of the two major clades of Plasmodium chabaudi PIRs across the blood cycle, and the apparent lack of expression on the red cell surface strongly suggest that the function(s) of this gene family may differ from those of other multigene families of Plasmodium, such as the var genes of Plasmodium falciparum.

The efficient delivery of RNA molecules to restore the expression of a missing or inadequately functioning protein in a target cell and the intentional specific modification of the host genome using engineered nucleases represent therapeutic concepts that are revolutionizing modern medicine. The initiation of several clinical trials using these approaches to treat metabolic liver disorders as well as the recently reported remarkable results obtained by patients with transthyretin amyloidosis highlight the advances in this field and show the potential of these therapies to treat these diseases safely and efficaciously. These advances have been possible due, firstly, to significant improvements made in RNA chemistry that increase its stability and prevent activation of the innate immune response and, secondly, to the development of very efficient liver‐targeted RNA delivery systems. In parallel, the breakout of CRISPR/CRISPR‐associated 9–based technology in the gene editing field has marked a turning point in in vivo modification of the cellular genome with therapeutic purposes, which can be based on gene supplementation, correction, or silencing. In the coming years we are likely to witness the therapeutic potential of these two strategies both separately and in combination. In this review we summarize the preclinical data obtained in animal models treated with mRNA as a therapeutic agent and discuss the different gene editing strategies applied to the treatment of liver diseases, highlighting both their therapeutic efficacy as well as safety concerns.

49Development of advanced gene and cell therapies for the treatment of genetic diseases requires 50 confident animal and cellular models to test their efficacy and is crucial in the cases where no 51 primary samples from patients are available. CRISPR/Cas9 technology, has become one of the 52 most spread endonuclease tools for editing the genome at will. Moreover, it is known that the use 53 of two guides tends to produce the precise deletion between the guides via NHEJ. Different 54 distances between guides were tested (from 8 to 500 base pairs). We found that distances 55 between the two cutting sites and orientation of Cas9 protein-DNA interaction are important for 56 the efficiency within the optimal range (30-60 bp), we could obtain new genetically reproducible 57 models for two rare disease, a Pyruvate Kinase Deficiency model, using human primary cells, and a 58 (for in vivo primary hyperoxaluria therapeutic mouse model. We demonstrate that the use of a 2-59 guideRNAs at the optimal distance and orientation is a powerful strategy for disease modelling in 60 those diseases where the availability of primary cells is limited. 61 62 63 64 65 standard chow and water. Agxt1-/-mice were genotyped as described (Salido et al. 2006). Age-127 matched C57BL/6J mice (Harlan laboratories) were used as control animals. A final dose of 1x10 13 128 vg/kg (5x10 12 vg/kg/vector) was administered to 12-14-week-old Agxt1-/-male animals by 129 intravenous injection. Animals were sacrificed and livers harvested 21 days after the 130 administration of the vectors. 131 132 133