Lucia Susani

There are thousands of rare genetic diseases that could be treated with classical gene therapy strategies such as the addition of the defective gene via viral or non-viral delivery or by direct gene editing. However, several genetic defects are too complex for these approaches. These “genomic mutations” include aneuploidies, intra and inter chromosomal rearrangements, large deletions, or inversion and copy number variations. Chromosome transplantation (CT) refers to the precise substitution of an endogenous chromosome with an exogenous one. By the addition of an exogenous chromosome and the concomitant elimination of the endogenous one, every genetic defect, irrespective of its nature, could be resolved. In the current review, we analyze the state of the art of this technique and discuss its possible application to human pathology. CT might not be limited to the treatment of human diseases. By working on sex chromosomes, we showed that female cells can be obtained from male cells, since chromosome-transplanted cells can lose either sex chromosome, giving rise to 46,XY or 46,XX diploid cells, a modification that could be exploited to obtain female gametes from male cells. Moreover, CT could be used in veterinary biology, since entire chromosomes containing an advantageous locus could be transferred to animals of zootechnical interest without altering their specific genetic background and the need for long and complex interbreeding. CT could also be useful to rescue extinct species if only male cells were available. Finally, the generation of “synthetic” cells could be achieved by repeated CT into a recipient cell. CT is an additional tool for genetic modification of mammalian cells.

There are thousands of rare genetic diseases that could be treated with classical gene therapy strategies such as the addition of the defective gene via viral or non-viral delivery or by direct gene editing. However, several genetic defects are too complex for these approaches. These “genomic mutations” include aneuploidies, intra and inter chromosomal rearrangements, large deletions, or inversion and copy number variations. Chromosome Transplantation (CT) refers to the precise substitution of an endogenous chromosome with an exogenous one. By the addition of an exogenous chromosome and the concomitant elimination of the endogenous one, every genetic defect, irrespective of its nature, could be resolved. In the current review, we analyze the state of the art of this technique and discuss its possible application to human pathology. CT might not be limited to the treatment of human diseases. By working on sex chromosomes, we showed that female cells can be obtained from male cells, since chromosome-transplanted cells can lose either sex chromosome, giving rise to 46,XY or 46,XX diploid cells, a modification that could be exploited to obtain female gametes from male cells. Moreover, CT could be used in veterinary biology, since entire chromosomes containing an advantageous locus could be transferred to animals of zootechnical interest without altering their specific genetic background and the need for long and complex interbreeding. CT could also be useful to rescue extinct species if only male cells were available. Finally, the generation of “synthetic” cells could be achieved by repeated CT into a recipient cell. CT is an additional tool for genetic modification of mammalian cells.

In spite of the progress in gene editing achieved in recent years, a subset of genetic diseases involving structural chromosome abnormalities, including aneuploidies, large deletions and complex rearrangements, cannot be treated with conventional gene therapy approaches. We have previously devised a strategy, dubbed chromosome transplantation (CT), to replace an endogenous mutated chromosome with an exogenous normal one. To establish a proof of principle for our approach, we chose as disease model the chronic granulomatous disease (CGD), an X-linked severe immunodeficiency due to abnormalities in CYBB (GP91) gene, including large genomic deletions. We corrected the gene defect by CT in induced pluripotent stem cells (iPSCs) from a CGD male mouse model. The Hprt gene of the endogenous X chromosome was inactivated by CRISPR/Cas9 technology thus allowing the exploitation of the hypoxanthine–aminopterin–thymidine selection system to introduce a normal donor X chromosome by microcell-mediated chromosome transfer. X-transplanted clones were obtained, and diploid XY clones which spontaneously lost the endogenous X chromosome were isolated. These cells were differentiated toward the myeloid lineage, and functional granulocytes producing GP91 protein were obtained. We propose the CT approach to correct iPSCs from patients affected by other X-linked diseases with large deletions, whose treatment is still unsatisfactory. Stem Cells 2019;37:876–887

CRISPR-Cas9 technology is a relatively recently developed tool for easy and efficient targeting of DNA. However, its efficiency for the repair of a mutated sequence is low. Moreover, most CRISPR-based gene correction approaches require the use of an exogenous template. Here, we investigated whether we could use the CRISPR-Cas9 system and the autologous repair machinery to correct human recessive genetic disorders having two different mutations in two alleles (compound heterozygotes). We reasoned that by targeting an intronic sequence located between the two mutations, we could generate at least one normal allele via the repair of induced double-strand breaks through either gene conversion or mitotic crossover. In particular, using a simple hypoxanthine-guanine phosphoribosyltransferase (Hprt)-based system, we show we can form a normal and functional Hprt gene. Thus, we give proof of principle that homology-directed recombination can be exploited in compound heterozygote cells to correct a genetic defect without exogenous templates.

Autosomal recessive osteopetroses (AROs) are rare, genetically heterogeneous skeletal diseases with increased bone density that are often lethal if left untreated. A precise molecular classification is relevant for the patient's management, because in some subgroups hematopoietic stem cell transplantation (HSCT), which is the only curative therapy, is contraindicated. In two unrelated ARO patients, the molecular analysis revealed the presence of a synonymous variant in known ARO genes, namely in the TCIRG1 gene in one patient and in the CLCN7 in the other patient, predicted to impact on the splicing process. In the latter case, sequencing of the transcript confirmed the splicing defect, whereas in the former, for whom an RNA sample was not available, the defect was reconstructed in vitro by the minigene technology. These results strongly suggest that these synonymous changes were responsible for the disease in our patients. Our findings are novel with respect to ARO and add to the few reports in literature dealing with different diseases, underlining the importance of cDNA analysis for the correct assessment of exonic changes, even when exome sequencing is performed. In particular, we highlight the possibility that at least in some cases ARO is due to synonymous changes, erroneously considered clinically silent, in the genes already described in literature, and suggest carefully reevaluating the sequencing results of these genes when mutations are not found at a first analysis. In addition, with respect to the CLCN7 gene, we suggest that synonymous variants might also contribute to the large spectrum of severity typical of CLCN7-dependent osteopetrosis through more subtle, but not negligible, effects on protein availability and functionality. © 2016 American Society for Bone and Mineral Research.

Chromosome transplantation as a novel approach for correcting complex genomic disorders

The clustered regularly interspaced short palindromic repeat (CRISPR)/associated 9 (Cas9) technology has been recently added to the tools allowing efficient and easy DNA targeting, representing a very promising approach to gene engineering. Using the CRISPR/Cas9 system we have driven the integration of exogenous DNA sequences to the X-linked Hprt gene of mouse embryonic stem cells. We show here that a simple fluorescence in situ hybridization (FISH)-based strategy allows the detection and the frequency evaluation of non-specific integrations of a given plasmid. FISH analysis revealed that these integrations do not match the software predicted off-target loci. We conclude that the frequency of these CRISPR-mediated off-target DNA cuts is negligible, since, due to the occurrence of spontaneous double-strand breaks, we observed more aspecific plasmid integrations than those corresponding to predicted off-target sites.

Mutations in the TCIRG1 gene, coding for a subunit of the osteoclast proton pump, are responsible for more than 50% of cases of human malignant autosomal recessive osteopetrosis (ARO), a rare inherited bone disease with increased bone density owing to a failure in bone resorption. A wide variety of mutations has been described, including missense, nonsense, small deletions/insertions, splice‐site mutations, and large genomic deletions, all leading to a similar severe presentation. So far, to the best of our knowledge, no report of a mild phenotype owing to recessive TCIRG1 mutations is present neither in our series of more than 100 TCIRG1‐dependent ARO patients nor in the literature. Here we describe an 8‐year‐old patient referred to us with a clinical diagnosis of ARO, based on radiological findings; of note, no neurological or hematological defects were present in this girl. Surprisingly, we identified a novel nucleotide change in intron 15 of the TCIRG1 gene at the homozygous state, leading to the production of multiple aberrant transcripts, but also, more importantly, of a limited amount of the normal transcript. Our results show that a low level of normal TCIRG1 protein can dampen the clinical presentation of TCIRG1‐dependent ARO. On this basis, a small amount of protein might be sufficient to rescue, at least partially, the severe ARO phenotype, and this is particularly important when gene therapy approaches are considered. In addition, we would also recommend that the TCIRG1 gene be included in the molecular diagnosis of mild forms of human ARO. © 2014 Italian National Research Council. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Human Autosomal Recessive Osteopetrosis (ARO) is a genetically heterogeneous disorder caused by reduced bone resorption by osteoclasts. In 2000, we found that mutations in the TCIRG1 gene encoding for a subunit of the proton pump (V‐ATPase) are responsible for more than one‐half of ARO cases. Since then, five additional genes have been demonstrated to be involved in the pathogenesis of the disease, leaving approximately 25% of cases that could not be associated with a genotype. Very recently, a mutation in the sorting nexin 10 (SNX10) gene, whose product is suggested to interact with the proton pump, has been found in 3 consanguineous families of Palestinian origin, thus adding a new candidate gene in patients not previously classified. Here we report the identification of 9 novel mutations in this gene in 14 ARO patients from 12 unrelated families of different geographic origin. Interestingly, we define the molecular defect in three cases of “Västerbottenian osteopetrosis,” named for the Swedish Province where a higher incidence of the disease has been reported. In our cohort of more than 310 patients from all over the world, SNX10‐dependent ARO constitutes 4% of the cases, with a frequency comparable to the receptor activator of NF‐κB ligand (RANKL), receptor activator of NF‐κB (RANK) and osteopetrosis‐associated transmembrane protein 1 (OSTM1)‐dependent subsets. Although the clinical presentation is relatively variable in severity, bone seems to be the only affected tissue and the defect can be almost completely rescued by hematopoietic stem cell transplantation (HSCT). These results confirm the involvement of the SNX10 gene in human ARO and identify a new subset with a relatively favorable prognosis as compared to TCIRG1‐dependent cases. Further analyses will help to better understand the role of SNX10 in osteoclast physiology and verify whether this protein might be considered a new target for selective antiresorptive therapies. © 2013 American Society for Bone and Mineral Research.