530. Development of New Lentiviral Vectors With a Reduced Splicing Interference Potential and a Safer In Vivo Genotoxic Profile

The excellent therapeutic potential of self-inactivating (SIN) lentiviral vectors (LV) has been demonstrated in pre-clinical studies and clinical trials. However, weaker mechanisms of insertional mutagenesis could endanger their clinical applications. Systemic vector injection into newborn tumor-prone Cdkn2a-/- and Cdkn2a+/-mice, conducted in our previous work, demonstrated that SINLVs harboring strong or moderate enhancer/promoters in internal position caused acceleration in hematopoietic tumor onset with respect to control mice. Integration sites analyses of vector-induced tumor showed that oncogene activations or tumor suppressor inactivation by LV integrations occur by combining mechanisms of transcript truncation, induction of aberrant splicing and/or enhancer-mediated overexpression of cellular transcription units. Although oncogene activation may be reduced by the use of self-inactivating design, moderate cellular promoters and insulator sequences how to reduce genotoxic splicing-capture events and aberrant transcript formation triggered by vector integration is still unclear.From this and a previous study, we identified the LV sequences most frequently involved in chimeric transcript formation. In our rationale, these LV sequences could be tagged by sequences complementary to microRNAs (mirT sequence) active in hematopoietic cells in order to allow selective degradation, through the miRNA pathway, of vector-mediated aberrantly spliced transcripts. Hence, we specifically designed SIN LVs harboring mirT sequences recognized by mir223 and mir142-3p (that are expressed in hematopoietic lineages) within the SIN LTR (mirsT-LTR LV) or in the vector backbone and outside the gene expression cassette (mirT LV). We then assessed the genotoxicity of the SIN LVs harboring mirT sequences by taking advantage of our in vivo models. Interestingly, injection of mirsT-LTR LV (N=73) and mirT LV (N=73) in Cdkn2a-/- mice did not caused any significant acceleration in hematopoietic tumor onset compared to control un-injected mice (N=40). Similar results have been obtained after injection in Cdkn2a+/- mice (N=28 for mirsT-LTR LV, N=26 for mirT LV and N=34 un-injected mice). We are currently performing integration site analyses in Cdkn2a-/- and Cdkn2a+/- treated mice to dissect if and how the integrated mirsT-LTR LV and mirT LV proviral genome interacts with the surrounding cellular genome.Overall, these studies show that this new advanced design lentiviral vectors completely abrogated residual vector genotoxicity in highly sensitive mouse models and could represent the vector design of choice in future gene therapy applications

529. Lentiviral Vectors with a Reduced Splicing Interference Potential Have a Significantly Improved Safety Profile In Vivo

Genotoxicity assays based on systemic vector injection into newborn tumor-prone Cdkn2a−/− and Cdkn2a+/− mice has shown that self-inactivating (SIN) lentiviral vector (LV) harboring strong or moderate enhancer/promoters in internal position caused acceleration in hematopoietic tumor onset compared to control mice. Integration site (IS) analysis in vector-induced tumors showed that oncogene activation or tumor suppressor inactivation occurs by mechanisms of aberrant splicing and/or enhancer-mediated overexpression of cellular genes. Although oncogene activation may be reduced by the use of SIN design, moderate cellular promoters and insulator sequences, how to reduce genotoxic splicing-capture events and aberrant transcript formation triggered by vector integration is still unclear. Here, we specifically designed SINLVs harboring sequences complementary to microRNAs (mirT sequence) which are active in hematopoietic cells (mir223 and mir142-3p) within the SIN LTR (mirsT-LTR.LV) or in the vector backbone and outside the gene expression cassette (mirT-LV). In our rationale, the mirT sequences when incorporated in an aberrantly generated mRNA would be selectively degraded through the miRNA pathway. Thus, by taking advantage of our in vivo models, we assessed the genotoxicity of these LVs with mirT sequences. Systemic injection of mirsT-LTR.LV (N=34) and mirT-LV (N=39) in Cdkn2a−/− mice did not cause any significant acceleration in hematopoietic tumor onset compared to un-injected mice (N=37) or mice injected with a SINLV that does not harbor mirT sequences (N=24). Similar results have been obtained after injection of the same vectors in Cdkn2a+/− mice (N=29 mirsT-LTR.LV, N=25 mirT-LV, N=40 un-injected and N=15 injected control mice). To gain additional information on the safety profile of these vectors, we performed IS analysis (N>10,000) in tumor-derived DNA. By this analysis, we previously found that Map3k8 activation by LV insertions was the major mechanism of genotoxicity when prototypical SINLVs were injected into Cdkn2a−/− mice. Now, we found that mice treated with mirsT-LTR.LV and mirT-LV did not show any Map3k8 activating insertions, suggesting that the new vectors are efficient in preventing its activation and confirming their superior safety profile. Furthermore, as expected, Pten was the most frequently targeted gene in tumors derived from Cdkn2a−/− mice injected with the LVs harboring mirT sequences. Pten insertions mainly targeted exons, suggesting the potential inactivation of its transcription unit. Finally, we found that Sfi1 was the major Common Insertion Site (CIS) in Cdkn2a+/− mice injected with LVs harboring mirT sequences. This CIS gene however appears to be the product of an intrinsic bias of LV integration, rather than the result of a selection process. Overall, our studies showed that these new advanced design LVs have a significantly improved safety profile and could represent the vector design of choice in future gene therapy applications

3. Safety Assessment of SIN LVs Harboring Chromatin Insulators in the Sensitive Cdkn2a-/- In Vivo Genotoxicity Assay Show Enhancer-Blocking Activity of Specific Insulator Sequences

Chromatin insulators (CI) have been proposed as safety features to increase the safety of self-inactivating (SIN) lentiviral vectors (LV) for gene therapy applications.By taking advantage of an in vivo genotoxicity assay based on the systemic injection of LVs in newborn tumor-prone Cdkn2a-/- mice we were able to measure vector-induced genotoxicity as an accelerated tumor onset that was proportional to the genotoxic potential of the tested LV. Importantly, we took advantage of integration sites (IS) analysis to qualitatively characterize CI that were shown by other in vitro and ex vivo studies to function as insulators. Recently we showed for the first time that a CAAT-box binding Nuclear factor 1 (CTF/NF1)-based CI, when cloned in the LTRs of a SIN.LV with a strong SFFV enhancer-promoter in internal position, significantly reduced the frequency of tumors harboring integrations activating Map3k8 oncogene accompanied by a marked skewing towards tumors harboring inactivating insertions targeting Pten.Here by using this stringent in vivo genotoxicity assay and IS analysis in tumors we expanded our studies towards other CI sequences whose function is regulated by the binding of the CCCTC-binding factor (CTCF), the best characterized insulator protein in vertebrates.Each CTCF-based insulating cassette was cloned in the LTRs of a LV construct containing the SFFV promoter in internal position (CTCF.SIN.LVs) and injected in Cdkn2a-/- mice. Interestingly, mice treated with some of the CTCF.SIN.LVs tested displayed an increased median survival time (ranging from 193.5 to 214 days) compared to mice treated with the uninsulated parental SIN.LV (186 days). Importantly, our preliminary IS analysis in tumors (881 IS) showed that two CTCF.SIN.LVs did not target Map3k8 oncogene while Pten was often disrupted by exonic insertions, an escape genotoxicity mechanism on which CI cannot act.These data confirm that the inclusion of two novel CTCF-based CIs of human origin completely abrogated the formation of tumors caused by enhancer-mediated activation of an oncogene in vivo.The ability of these two new insulator elements to block the crosstalk between powerful vector enhancers and cellular regulatory elements increase the safety of SIN LVs and justify their prompt adoption in future gene therapy applications

731. Hematopoietic Stem Cell Gene Transfer and Integration Site Analysis in Tumor-Prone Mice Uncovers Low Genotoxicity of Lentiviral Vector Integration

Insertional mutagenesis represents a major hurdle to successful gene therapy and mandates for sensitive pre-clinical assays of genotoxicity. Cdkn2a|[minus]|/|[minus]| mice are defective for p53 and Rb pathways, and are susceptible to a broad range of cancer-triggering genetic lesions. We exploited the sensitivity of these tumor-prone mice to develop an in-vivo genotoxicity assay, based on transplantation of Cdkn2a|[minus]|/|[minus]| hematopoietic stem cells (HSC), treated or not with prototypical retroviral (RV) and lentiviral (LV) vectors. In our rationale if RV or LV treatment is genotoxic, then transplanted mice will show a significantly earlier tumor onset. The sensitivity of the model was shown by the ability to detect a vector dose-dependent acceleration in tumor onset in mice transplanted with RV-treated cells. Such acceleration, as in previous studies, is consequent to genetic lesions, produced by vector integration, that cooperate with the germ-line mutation, and is contingent on LTR activity

Neural stem cell transplantation in patients with progressive multiple sclerosis: an open-label, phase 1 study

Innovative pro-regenerative treatment strategies for progressive multiple sclerosis (PMS), combining neuroprotection and immunomodulation, represent an unmet need. Neural precursor cells (NPCs) transplanted in animal models of multiple sclerosis have shown preclinical efficacy by promoting neuroprotection and remyelination by releasing molecules sustaining trophic support and neural plasticity. Here we present the results of STEMS, a prospective, therapeutic exploratory, non-randomized, open-label, single-dose-finding phase 1 clinical trial (NCT03269071, EudraCT 2016-002020-86), performed at San Raffaele Hospital in Milan, Italy, evaluating the feasibility, safety and tolerability of intrathecally transplanted human fetal NPCs (hfNPCs) in 12 patients with PMS (with evidence of disease progression, Expanded Disability Status Scale >= 6.5, age 18-55 years, disease duration 2-20 years, without any alternative approved therapy). The safety primary outcome was reached, with no severe adverse reactions related to hfNPCs at 2-year follow-up, clearly demonstrating that hfNPC therapy in PMS is feasible, safe and tolerable. Exploratory secondary analyses showed a lower rate of brain atrophy in patients receiving the highest dosage of hfNPCs and increased cerebrospinal fluid levels of anti-inflammatory and neuroprotective molecules. Although preliminary, these results support the rationale and value of future clinical studies with the highest dose of hfNPCs in a larger cohort of patients

Colorectal Cancer Stage at Diagnosis Before vs During the COVID-19 Pandemic in Italy

IMPORTANCE Delays in screening programs and the reluctance of patients to seek medical attention because of the outbreak of SARS-CoV-2 could be associated with the risk of more advanced colorectal cancers at diagnosis. OBJECTIVE To evaluate whether the SARS-CoV-2 pandemic was associated with more advanced oncologic stage and change in clinical presentation for patients with colorectal cancer. DESIGN, SETTING, AND PARTICIPANTS This retrospective, multicenter cohort study included all 17 938 adult patients who underwent surgery for colorectal cancer from March 1, 2020, to December 31, 2021 (pandemic period), and from January 1, 2018, to February 29, 2020 (prepandemic period), in 81 participating centers in Italy, including tertiary centers and community hospitals. Follow-up was 30 days from surgery. EXPOSURES Any type of surgical procedure for colorectal cancer, including explorative surgery, palliative procedures, and atypical or segmental resections. MAIN OUTCOMES AND MEASURES The primary outcome was advanced stage of colorectal cancer at diagnosis. Secondary outcomes were distant metastasis, T4 stage, aggressive biology (defined as cancer with at least 1 of the following characteristics: signet ring cells, mucinous tumor, budding, lymphovascular invasion, perineural invasion, and lymphangitis), stenotic lesion, emergency surgery, and palliative surgery. The independent association between the pandemic period and the outcomes was assessed using multivariate random-effects logistic regression, with hospital as the cluster variable. RESULTS A total of 17 938 patients (10 007 men [55.8%]; mean [SD] age, 70.6 [12.2] years) underwent surgery for colorectal cancer: 7796 (43.5%) during the pandemic period and 10 142 (56.5%) during the prepandemic period. Logistic regression indicated that the pandemic period was significantly associated with an increased rate of advanced-stage colorectal cancer (odds ratio [OR], 1.07; 95%CI, 1.01-1.13; P = .03), aggressive biology (OR, 1.32; 95%CI, 1.15-1.53; P < .001), and stenotic lesions (OR, 1.15; 95%CI, 1.01-1.31; P = .03). CONCLUSIONS AND RELEVANCE This cohort study suggests a significant association between the SARS-CoV-2 pandemic and the risk of a more advanced oncologic stage at diagnosis among patients undergoing surgery for colorectal cancer and might indicate a potential reduction of survival for these patients

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Anales del III Congreso Internacional de Vivienda y Ciudad "Debate en torno a la nueva agenda urbana"

Acta de congresoEl III Congreso Internacional de Vivienda y Ciudad “Debates en torno a la NUEVa Agenda Urbana”, ha sido una apuesta de alto compromiso por acercar los debates centrales y urgentes que tensionan el pleno ejercicio del derecho a la ciudad. Para ello las instituciones organizadoras (INVIHAB –Instituto de Investigación de Vivienda y Hábitat y MGyDH-Maestría en Gestión y Desarrollo Habitacional-1), hemos convidado un espacio que se concretó con potencia en un debate transdisciplinario. Convocó a intelectuales de prestigio internacional, investigadores, académicos y gestores estatales, y en una metodología de innovación articuló las voces académicas con las de las organizaciones sociales y/o barriales en el Foro de las Organizaciones Sociales que tuvo su espacio propio para dar voz a quienes están trabajando en los desafíos para garantizar los derechos a la vivienda y los bienes urbanos en nuestras ciudades del Siglo XXI