Targeted genome editing via engineered nucleases is an exciting area of biomedical research and holds potential for clinical applications. Despite rapid advances in the field, in vivo targeted transgene integration is still infeasible because current tools are inefficient1, especially for non-dividing cells, which compose most adult tissues. This poses a barrier for uncovering fundamental biological principles and developing treatments for a broad range of genetic disorders2. Based on clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9)3,4 technology, here we devise a homology-independent targeted integration (HITI) strategy, which allows for robust DNA knock-in in both dividing and non-dividing cells in vitro and, more importantly, in vivo (for example, in neurons of postnatal mammals). As a proof of concept of its therapeutic potential, we demonstrate the efficacy of HITI in improving visual function using a rat model of the retinal degeneration condition retinitis pigmentosa. The HITI method presented here establishes new avenues for basic research and targeted gene therapies.

Objectives:To evaluate the efficacy and safety of 18 months of tafamidis treatment in patients with early-stage V30M transthyretin familial amyloid polyneuropathy (TTR-FAP). Methods:In this randomized, double-blind trial, patients received tafamidis 20 mg QD or placebo.Coprimary endpoints were the Neuropathy Impairment Score-Lower Limbs (NIS-LL) responder analysis (Ͻ2-point worsening) and treatment-group difference in the mean change from baseline in Norfolk Quality of Life-Diabetic Neuropathy total score (TQOL) in the intent-to-treat (ITT) population (n ϭ 125). These endpoints were also evaluated in the efficacy-evaluable (EE; n ϭ 87) population. Secondary endpoints, including changes in neurologic function, nutritional status, and TTR stabilization, were analyzed in the ITT population.Results: There was a higher-than-anticipated liver transplantation dropout rate. No differences were observed between the tafamidis and placebo groups for the coprimary endpoints, NIS-LL responder analysis (45.3% vs 29.5% responders; p ϭ 0.068) and change in TQOL (2.0 vs 7.2; p ϭ 0.116) in the ITT population. In the EE population, significantly more tafamidis patients than placebo patients were NIS-LL responders (60.0% vs 38.1%; p ϭ 0.041), and tafamidis patients had better-preserved TQOL (0.1 vs 8.9; p ϭ 0.045). Significant differences in most secondary endpoints favored tafamidis. TTR was stabilized in 98% of tafamidis and 0% of placebo patients (p Ͻ 0.0001). Adverse events were similar between groups. Conclusions:Although the coprimary endpoints were not met in the ITT population, tafamidis was associated with no trend toward more NIS-LL responders and a significant reduction in worsening of most neurologic variables, supporting the hypothesis that preventing TTR dissociation can delay peripheral neurologic impairment. Classification of evidence:This study provides Class II evidence that 20 mg tafamidis QD was associated with no difference in clinical progression in patients with TTR-FAP, as measured by the NIS-LL and the Norfolk QOL-DN score. Secondary outcomes demonstrated a significant delay in peripheral neurologic impairment with tafamidis, which was well tolerated over 18 months. Neurology® 2012;79:785-792 GLOSSARY AE ϭ adverse event; ANCOVA ϭ analysis of covariance; ARR ϭ absolute risk reduction; CI ϭ confidence interval; DPN ϭ diabetic polyneuropathy; EE ϭ efficacy-evaluable; ITT ϭ intent-to-treat; LS Mean ϭ least-squares mean; mBMI ϭ modified body mass index; NIS-LL ϭ Neuropathy Impairment Score-Lower Limbs; NNT ϭ number needed to treat; QOL ϭ quality of life; QOL-DN ϭ Quality of Life-Diabetic Neuropathy Questionnaire; TQOL ϭ total quality of life; TTR-FAP ϭ transthyretin familial amyloid polyneuropathy.

SUMMARY Aging is the major risk factor for many human diseases. In vitro studies have demonstrated that cellular reprogramming to pluripotency reverses cellular age, but alteration of the aging process through reprogramming has not been directly demonstrated in vivo. Here, we report that partial reprogramming by short-term cyclic expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) ameliorates cellular and physiological hallmarks of aging and prolongs lifespan in a mouse model of premature aging. Similarly, expression of OSKM in vivo improves recovery from metabolic disease and muscle injury in older wild-type mice. The amelioration of age-associated phenotypes by epigenetic remodeling during cellular reprogramming highlights the role of epigenetic dysregulation as a driver of mammalian aging. Establishing in vivo platforms to modulate age-associated epigenetic marks may provide further insights into the biology of aging.

At 2 years, SRL conversion among patients with baseline GFR more than 40 mL/min was associated with excellent patient and graft survival, no difference in BCAR, increased urinary protein excretion, and a lower incidence of malignancy compared with CNI continuation. Superior renal function was observed among patients who remained on SRL through 12 to 24 months, particularly in the subgroup of patients with baseline GFR more than 40 mL/min and UPr/Cr less than or equal to 0.11.

SUMMARY Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.

BACKGROUND: Transplant recipients in whom cutaneous squamous-cell carcinomas develop are at high risk for multiple subsequent skin cancers. Whether sirolimus is useful in the prevention of secondary skin cancer has not been assessed. METHODS: In this multicenter trial, we randomly assigned transplant recipients who were taking calcineurin inhibitors and had at least one cutaneous squamouscell carcinoma either to receive sirolimus as a substitute for calcineurin inhibitors (in 64 patients) or to maintain their initial treatment (in 56). The primary end point was survival free of squamous-cell carcinoma at 2 years. Secondary end points included the time until the onset of new squamous-cell carcinomas, occurrence of other skin tumors, graft function, and problems with sirolimus. RESULTS: Survival free of cutaneous squamous-cell carcinoma was significantly longer in the sirolimus group than in the calcineurin-inhibitor group. Overall, new squamous-cell carcinomas developed in 14 patients (22%) in the sirolimus group (6 after withdrawal of sirolimus) and in 22 (39%) in the calcineurin-inhibitor group (median time until onset, 15 vs. 7 months; P=0.02), with a relative risk in the sirolimus group of 0.56 (95% confidence interval, 0.32 to 0.98). There were 60 serious adverse events in the sirolimus group, as compared with 14 such events in the calcineurin-inhibitor group (average, 0.938 vs. 0.250). There were twice as many serious adverse events in patients who had been converted to sirolimus with rapid protocols as in those with progressive protocols. In the sirolimus group, 23% of patients discontinued the drug because of adverse events. Graft function remained stable in the two study groups. CONCLUSIONS: Switching from calcineurin inhibitors to sirolimus had an antitumoral effect among kidney-transplant recipients with previous squamous-cell carcinoma. These observations may have implications concerning immunosuppressive treatment of patients with cutaneous squamous-cell carcinomas. (Funded by Hospices Civils de Lyon and others; TUMORAPA ClinicalTrials.gov number, NCT00133887.).

Post-transplant malignancy is recognised as being a major limitation to the success of solid organ transplantation and it is currently considered one of the unavoidable costs of long-term immunosuppressive therapy. However, the continual introduction of new immunosuppressive drugs and the growing knowledge about their different oncogenic profiles, requires a continuous evaluation of the available evidence on this topic. The incidence and risk of malignancy is elevated in solid organ transplant recipients compared with the general population. As proof of the relationship between immunosuppressive therapy and post-transplant malignancy, epidemiological data reveal that the length of exposure to immunosuppressive therapy and the intensity of therapy are clearly related to the post-transplant risk of malignancy, and that once cancer has developed, more intense immunosuppression can translate into more aggressive tumour progression in terms of accelerated growth and metastasis and lower patient survival. The association between malignancy and immunosuppressive therapy is mediated through several pathogenic factors. Indirectly, immunosuppressive drugs greatly increase the post-transplant risk of malignancy by impairing cancer surveillance and facilitating the action of oncogenic viruses. However, the direct pro- and anti-oncogenic actions of immunosuppressants also play an important role. The cancer-promoting effect of calcineurin inhibitors, independently of depressed immunosurveillance, has been demonstrated in recent years, and currently only mammalian target of rapamycin (mTOR) inhibitors have shown simultaneous immunosuppressive and antitumour properties. Reports of the initial results of the reduced incidence of cancer in organ transplant recipients receiving mTOR inhibitor therapy strongly indicate separate pathways for pharmacological immunosuppression and oncogenesis. The role of mTOR inhibitors has been firmly established for the treatment of post-transplant Kaposi's sarcoma and its role in the management of patients with other post-transplant malignancies should be clarified as soon as possible. Prevention of morbidity and mortality resulting from post-transplant malignancy should become a main endpoint in solid organ transplant programmes, and the choice and management of immunosuppressive therapy in each phase of transplantation plays a central role in this objective. Although comprehensive and rigorous information about the management of immunosuppressive therapy in transplant recipients at risk of or affected by cancer is still lacking, new experimental and clinical data about mTOR inhibitors offers novel approaches to this problem.

Patient and graft survival and the incidence of biopsy-proven acute rejection at 12 months were comparable between sirolimus and CsA, whereas safety profiles were different. These data suggest that sirolimus may be used as primary therapy for the prevention of acute rejection.