Cancer-associated POT1 mutations lead to telomere elongation without induction of a DNA damage response.

Mutations in the shelterin protein POT1 are associated with chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, angiosarcoma, melanoma, and other cancers. These cancer-associated POT1 (caPOT1) mutations are generally heterozygous, missense, or nonsense mutations occurring throughout the POT1 reading frame. Cancers with caPOT1 mutations have elongated telomeres and show increased genomic instability, but which of the two phenotypes promotes tumorigenesis is unclear. We tested the effects of CAS9-engineered caPOT1 mutations in human embryonic and hematopoietic stem cells (hESCs and HSCs, respectively). HSCs with caPOT1 mutations did not show overt telomere damage. In vitro and in vivo competition experiments showed the caPOT1 mutations did not confer a selective disadvantage. Since DNA damage signaling is known to affect the fitness of HSCs, the data argue that caPOT1 mutations do not cause significant telomere damage. Furthermore, hESC lines with caPOT1 mutations showed no detectable telomere damage response while showing consistent telomere elongation. Thus, caPOT1 mutations are likely selected for during cancer progression because of their ability to elongate telomeres and extend the proliferative capacity of the incipient cancer cells.

Editing TINF2 as a potential therapeutic approach to restore telomere length in dyskeratosis congenita.

Mutations in the TINF2 gene, encoding the shelterin protein TIN2, cause telomere shortening and the inherited bone marrow (BM) failure syndrome dyskeratosis congenita (DC). A lack of suitable model systems limits the mechanistic understanding of telomere shortening in the stem cells and thus hinders the development of treatment options for BM failure. Here, we endogenously introduced TIN2-DC mutations in human embryonic stem cells (hESCs) and human hematopoietic stem and progenitor cells (HSPCs) to dissect the disease mechanism and identify a gene-editing strategy that rescued the disease phenotypes. The hESCs with the T284R disease mutation exhibited the short telomere phenotype observed in DC patients. Yet, telomeres in mutant hESCs did not trigger DNA damage responses at telomeres or show exacerbated telomere shortening when differentiated into telomerase-negative cells. Disruption of the mutant TINF2 allele by introducing a frameshift mutation in exon 2 restored telomere length in stem cells and the replicative potential of differentiated cells. Similarly, we introduced TIN2-DC disease variants in human HSPCs to assess the changes in telomere length and proliferative capacity. Lastly, we showed that editing at exon 2 of TINF2 that restored telomere length in hESCs could be generated in TINF2-DC patient HSPCs. Our study demonstrates a simple genetic intervention that rescues the TIN2-DC disease phenotype in stem cells and provides a versatile platform to assess the efficacy of potential therapeutic approaches in vivo.

CD90-targeted lentiviral vectors for HSC gene therapy.

Hematopoietic stem cell (HSC) gene therapy is currently performed on CD34+ hematopoietic stem and progenitor cells containing less than 1% true HSCs and requiring a highly specialized infrastructure for cell manufacturing and transplantation. We have previously identified the CD34+CD90+ subset to be exclusively responsible for short- and long-term engraftment. However, purification and enrichment of this subset is laborious and expensive. HSC-specific delivery agents for the direct modification of rare HSCs are currently lacking. Here, we developed novel targeted viral vectors to specifically transduce CD90-expressing HSCs. Anti-CD90 single chain variable fragments (scFvs) were engineered onto measles- and VSV-G-pseudotyped lentiviral vectors that were knocked out for native targeting. We further developed a custom hydrodynamic titration methodology to assess the loading of surface-engineered capsids, measure antigen recognition of the scFv, and predict the performance on cells. Engineered vectors formed with minimal impairment in the functional titer, maintained their ability to fuse with the target cells, and showed highly specific recognition of CD90 on cells ex vivo. Most important, targeted vectors selectively transduced human HSCs with secondary colony-forming potential. Our novel HSC-targeted viral vectors have the potential to significantly enhance the feasibility of ex vivo gene therapy and pave the way for future in vivo applications.

Choosing the right mouse model: comparison of humanized NSG and NBSGW mice for in vivo HSC gene therapy.

ABSTRACT: In vivo hematopoietic stem cell (HSC) gene therapy is an emerging and promising area of focus in the gene therapy field. Humanized mouse models are frequently used to evaluate novel HSC gene therapy approaches. Here, we comprehensively evaluated 2 mouse strains, NSG and NBSGW. We studied human HSC engraftment in the bone marrow (BM), mobilization of BM-engrafted HSCs into circulation, in vivo transduction using vesicular stomatitis virus glycoprotein-pseudotyped lentiviral vectors (VSV-G LVs), and the expression levels of surface receptors needed for transduction of viral vectors. Our findings reveal that the NBSGW strain exhibits superior engraftment of human long-term HSCs compared with the NSG strain. However, neither model resulted in a significant increase in circulating human HSCs after mobilization. We show that time after humanization as well as human chimerism levels and platelet counts in the peripheral blood can be used as surrogates for human HSC engraftment in the BM. Furthermore, we observed low expression of the low-density lipoprotein receptor, a requirement for VSV-G LV transduction, in the human HSCs present in the murine BM. Our comprehensive characterization of humanized mouse models highlights the necessity of proper validation of the model and methods to study in vivo HSC gene therapy strategies.

TINF2 is a haploinsufficient tumor suppressor that limits telomere length.

Telomere shortening is a presumed tumor suppressor pathway that imposes a proliferative barrier (the Hayflick limit) during tumorigenesis. This model predicts that excessively long somatic telomeres predispose to cancer. Here, we describe cancer-prone families with two unique TINF2 mutations that truncate TIN2, a shelterin subunit that controls telomere length. Patient lymphocyte telomeres were unusually long. We show that the truncated TIN2 proteins do not localize to telomeres, suggesting that the mutations create loss-of-function alleles. Heterozygous knock-in of the mutations or deletion of one copy of TINF2 resulted in excessive telomere elongation in clonal lines, indicating that TINF2 is haploinsufficient for telomere length control. In contrast, telomere protection and genome stability were maintained in all heterozygous clones. The data establish that the TINF2 truncations predispose to a tumor syndrome. We conclude that TINF2 acts as a haploinsufficient tumor suppressor that limits telomere length to ensure a timely Hayflick limit.

Slo1 is the principal potassium channel of human spermatozoa.

Mammalian spermatozoa gain competence to fertilize an oocyte as they travel through the female reproductive tract. This process is accompanied by an elevation of sperm intracellular calcium and a membrane hyperpolarization. The latter is evoked by K(+) efflux; however, the molecular identity of the potassium channel of human spermatozoa (hKSper) is unknown. Here, we characterize hKSper, reporting that it is regulated by intracellular calcium but is insensitive to intracellular alkalinization. We also show that human KSper is inhibited by charybdotoxin, iberiotoxin, and paxilline, while mouse KSper is insensitive to these compounds. Such unique properties suggest that the Slo1 ion channel is the molecular determinant for hKSper. We show that Slo1 is localized to the sperm flagellum and is inhibited by progesterone. Inhibition of hKSper by progesterone may depolarize the spermatozoon to open the calcium channel CatSper, thus raising [Ca(2+)] to produce hyperactivation and allowing sperm to fertilize an oocyte. DOI:http://dx.doi.org/10.7554/eLife.01009.001.