Are human V\u3b42pos T cells really resistant to aging and Human Cytomegalovirus infection?

In their recent paper, Weili Xu et al. [1] described the different behaviors of V\u3b41pos and V\u3b42pos T cell subsets in response to lifelong stress and claimed that V\u3b42pos T cells are not affected by aging and Human Cytomegalovirus (HCMV) infection. While we agree that these two \u3b3\u3b4 T cell subsets diverge both in phenotype/function and in tissue distribution, we are somewhat surprised that authors did not take into account the several previously published and contradictory experimental evidence in regards to senescence of V\u3b42pos T cells [2,3]. These latter studies reported that HCMV infection not only induces a clonal expansion of a distinct V\u3b39neg/V\u3b42pos T cell subset, but also determines a concomitant adaptive differentiation from CD27high na\uefve cells to CD27low/neg terminal-effectors. However, Weili Xu et al. argued that the expression and kinetics of both CD27 and CD45RA surface markers do not change and follow the homeostatic changes of V\u3b42pos T cells. This statement goes in the opposite direction to previously reported findings as the CD27/CD45RA phenotype has been shown to mark the maturation and differentiation (TNa\uefve, TCentral-Memory, Teffector-Memory and TEffectory-Memory RA) of V\u3b42pos T cells. Indeed, the different surface expression of both CD27 and CD45 parallel the progressive decrease of telomere length, the proliferative capacity as well as the different effector-functions and resistance to death of V\u3b42+ T cells in response to antigens and homeostatic cytokines [4,5]. Hence, we believe that these controversial issues require further discussion beyond the unilateral conclusion given by the study of Weili Xu et al

Gene Therapy For Inherited Blood Diseases, From Viral Vectors To Gene Editing

Twenty-five years ago, genetically modified bone marrow cells were administered for the first time to a child suffering from adenosine deaminase (ADA) deficiency, a rare disorder of the immune system. Since then, gene therapy has struggled to find its place in clinical medicine, amid a rollercoaster of successes and setbacks and hype and skepticism with little precedent in modern times. Recently, a series of authoritative clinical studies proved that transplantation of genetically modified hematopoietic stem cells can cure severe diseases like immunodeficiencies, hemoglobinopathies and metabolic diseases, contributing to transforming gene therapy into one of the hottest area of investment for the biotechnology and the pharma industry. The basic technology for the genetic modification of stem cells relies on retroviral vectors, and particularly on those derived from oncoretroviruses or lentiviruses, such as HIV-1. Integration of these vectors in the genome may, however, have undesired effects caused by insertional deregulation of gene expression at the transcriptional or post-transcriptional level. The occurrence of severe adverse events in several clinical trials involving the transplantation of stem cells genetically corrected with retroviral vectors showed that insertional mutagenesis is not just a theoretical event, and that retroviral transgenesis is associated with a finite risk of genotoxicity. Addressing these issues brought new basic knowledge on virus-host interactions and on the biology and dynamics of human somatic stem cells. More recently, a new generation of technology emerged, aimed at correcting the genome rather than replacing defective gene function. This technology relies on designer nucleases capable of generating double- or single-stranded breaks in genomic DNA, which are then repaired either by error-prone non homologous end-joining or by the more precise homologous recombination. This allows generating knock-out mutations or repairing genes with remarkable precision and efficiency in many cell types. At Genethon, we are using lentiviral vector technology to correct Wiskott-Aldrich syndrome, X-linked SCID, chronic granulomatous disease and sickle-cell disease, while developing CRISPR/Cas9-based genome editing for a number of applications

Developing gene and cell therapies for rare diseases: An opportunity for synergy between academia and industry

For the last 20 years, academic research has been the major, and often only, driving force behind the spectacular development of gene transfer technology for the therapy of rare genetic diseases. Investors and industry became eventually interested in gene and cell therapy, due to the success of a series of pioneering clinical trials that proved efficacy and safety of last-generation technology, and to favorable orphan drug legislation in both Europe and the United States. Developing this forms of therapy is however complex and requires skills and knowledge not necessary available to the industry, which is better placed to develop processes and products and put them on the market. Cooperation between academia and industry is an opportunity to de-risk innovative approaches and ensure a faster and more economical development of therapies for diseases with high unmet medical needs and low-profit expectations

Interactions between Retroviruses and the Host Cell Genome

Replication-defective retroviral vectors have been used for more than 25 years as a tool for efficient and stable insertion of therapeutic transgenes in human cells. Patients suffering from severe genetic diseases have been successfully treated by transplantation of autologous hematopoietic stem-progenitor cells (HSPCs) transduced with retroviral vectors, and the first of this class of therapies, Strimvelis, has recently received market authorization in Europe. Some clinical trials, however, resulted in severe adverse events caused by vector-induced proto-oncogene activation, which showed that retroviral vectors may retain a genotoxic potential associated to proviral integration in the human genome. The adverse events sparked a renewed interest in the biology of retroviruses, which led in a few years to a remarkable understanding of the molecular mechanisms underlying retroviral integration site selection within mammalian genomes. This review summarizes the current knowledge on retrovirus-host interactions at the genomic level, and the peculiar mechanisms by which different retroviruses, and their related gene transfer vectors, integrate in, and interact with, the human genome. This knowledge provides the basis for the development of safer and more efficacious retroviral vectors for human gene therapy

Designing lentiviral vectors for gene therapy of genetic diseases

Lentiviral vectors are the most frequently used tool to stably transfer and express genes in the context of gene therapy for monogenic diseases. The vast majority of clinical applications involves an ex vivo modality whereby lentiviral vectors are used to transduce autologous somatic cells, ob-tained from patients and re-delivered to patients after transduction. Examples are hematopoietic stem cells used in gene therapy for hematological or neurometabolic diseases or T cells for immunotherapy of cancer. We review the design and use of lentiviral vectors in gene therapy of monogenic diseases, with a focus on controlling gene expression by transcriptional or post-transcriptional mechanisms in the context of vectors that have already entered a clinical development phase

On the way to become a natural killer cell

Natural Killer (NK) cells are innate lymphocytes playing pivotal roles in host defense and immune-surveillance. The homeostatic modulation of germ-line encoded/non-rearranged activating and inhibitory NK cell receptors (NKRs) determines the capability of these innate lymphocytes to either spare "self" cells or to kill viral-infected, tumor-transformed and heterologous cell targets. However, despite being discovered more than 40 years ago, several aspects of NK cell biology remain unknown or are still being debated. In particular, our knowledge of human NK cell ontogenesis and differentiation is still in its infancy as the majority of our experimental evidence on this topic mainly comes from findings obtained in vitro or with animal models in vivo. Although both the generation and the maintenance of human NK cells are sustained by hematopoietic stem cells (HSCs), the precise site(s) of NK cell development are still poorly defined. Indeed, HSCs and hematopoietic precursors are localized in different anatomical compartments that also change their ontogenic commitments before and after birth as well as in aging. Currently, the main site of NK cell generation and maturation in adulthood is considered the bone marrow, where their interactions with stromal cells, cytokines, growth factors, and other soluble molecules support and drive maturation. Different sequential stages of NK cell development have been identified on the basis of the differential expression of specific markers and NKRs as well as on the acquisition of specific effector-functions. All these phenotypic and functional features are key in inducing and regulating homing, activation and tissue-residency of NK cells in different human anatomic sites, where different homeostatic mechanisms ensure a perfect balance between immune tolerance and immune-surveillance. The present review summarizes our current knowledge on human NK cell ontogenesis and on the related pathways orchestrating a proper maturation, functions, and distributions

Gene therapy of inherited skin adhesion disorders: a critical overview

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Chemotherapy accelerates immune-senescence and functional impairments of Vδ2pos T cells in elderly patients affected by liver metastatic colorectal cancer.

Human (gamma delta) γδ T cells are unconventional innate-like lymphocytes displaying a broad array of anti-tumor activities with promising perspectives in cancer immunotherapy. In this context, Vδ2pos T cells represent the preferential target of several immunotherapy protocols against solid tumors. However, the impact of both aging and chemotherapy (CHT) on Vδ2pos T cells is still unknown. The present study evaluates with multi-parametric flow cytometry the frequencies, terminal differentiation, senescence and effector-functions of peripheral blood and tumor infiltrating Vδ2pos T cells purified from liver metastases (CLM) of patients affected by colorectal cancer (CRC) compared to those of sex- and age-matched healthy donors. The peripheral blood of CLM patients underwent CHT is characterized by decreased amounts of Vδ2pos T cells showing a relative increase of terminally-differentiated CD27neg/CD45RApos (TEMRA) cells. The enrichment of this latter subset is associated with an increased expression of the senescent marker CD57. The acquisition of CD57 on TEMRA Vδ2pos T cells is also coupled with impairments in cytotoxicity and production of TNF-α and IFN-γ. These features resemble the acquisition of an immune-senescent profile by Vδ2pos T cells from CLM patients that received CHT, a phenomenon that is also associated with the loss of the co-stimulatory marker CD28 and with the induced expression of CD16. The group of CLM patients underwent CHT and older than 60 years old showed higher frequencies of CD57pos and TEMRA Vδ2pos T cells. Similar results were found for tumor infiltrating Vδ2pos T cell subset purified from CLM specimens of patients treated with CHT. The toxicity of CHT regimens also affects the homeostasis of Vδ2pos T cells by inducing higher frequencies of circulating CD57pos TEMRA subset in CLM underwent CHT and younger than 60 years old. Taken together, our data demonstrate that the enrichment of senescent Vδ2pos T cells in CLM patients is not only induced by patients' aging but also by the toxicity of CHT that further accelerates the accumulation of CD57pos TEMRA cells highly dysfunctional in their anti-tumor activities. These results are important to both predict the clinical outcome of CLM and to optimize those protocols of cell cancer immunotherapy employing unconventional Vδ2pos T cells

Innate Immune Responses in the Outcome of Haploidentical Hematopoietic Stem Cell Transplantation to Cure Hematologic Malignancies

In the context of allogeneic transplant platforms, human leukocyte antigen (HLA)-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) represents one of the latest and most promising curative strategies for patients affected by high-risk hematologic malignancies. Indeed, this platform ensures a suitable stem cell source immediately available for virtually any patents in need. Moreover, the establishment in recipients of a state of immunologic tolerance toward grafted hematopoietic stem cells (HSCs) remarkably improves the clinical outcome of this transplant procedure in terms of overall and disease free survival. However, the HLA-mismatch between donors and recipients has not been yet fully exploited in order to optimize the Graft vs. Leukemia effect. Furthermore, the efficacy of haplo-HSCT is currently hampered by several life-threatening side effects including the onset of Graft vs. Host Disease (GvHD) and the occurrence of opportunistic viral infections. In this context, the quality and the kinetic of the immune cell reconstitution (IR) certainly play a major role and several experimental efforts have been greatly endorsed to better understand and accelerate the post-transplant recovery of a fully competent immune system in haplo-HSCT. In particular, the IR of innate immune system is receiving a growing interest, as it recovers much earlier than T and B cells and it is able to rapidly exert protective effects against both tumor relapses, GvHD and the onset of life-threatening opportunistic infections. Herein, we review our current knowledge in regard to the kinetic and clinical impact of Natural Killer (NK), \u3b3\u3b4 and Innate lymphoid cells (ILCs) IRs in both allogeneic and haplo-HSCT. The present paper also provides an overview of those new therapeutic strategies currently being implemented to boost the alloreactivity of the above-mentioned innate immune effectors in order to ameliorate the prognosis of patients affected by hematologic malignancies and undergone transplant procedures

131. Targeted Genome Editing in Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations in the survival-motor-neuron 1 (SMN1) telomeric gene. Deficiencies in the ubiquitous SMN function affect multiple tissues and organs; however neuronal tissue is primarily sensitive, resulting in α-motor neuron degeneration in the ventral horn of the spinal cord with subsequent neuromuscular-junction dysfunction and proximal muscle weakness. The onset of disease and degree of severity are variable in patients and they are determined in part by multiple copies of the centromeric homologue SMN2 that inversely correlate with the phenotypic severity. Indeed, SMN2 gene mainly produces a truncated form SMNΔ7 by aberrant alternative splicing and a small amount (~10%) of the fully active full-length SMN, thus buffering the SMN deficiency. A potential strategy for treating SMA patients is to increase SMN levels in the affected tissues, hence gene therapy and modifiers of SMN2-alternative splicing have proved therapeutic efficacy in SMA animal models.In this study, we explored the possibility of applying targeted genome editing technology to the human SMN locus in order to revert the SMN2 sequence to a SMN1-like sequence that may undergo proper splicing under the the endogenous transcriptional control. The resulting correction would be permanent and lead to longlasting protein production in gene-edited cells. We used the streptococcus pyogenes Cas9-CRISPR system to target the SMN2 gene at different locations. Two main strategies were explored: i) SMN1_exon7 addition/correction by promoting homology-driven DNA repair, ii) SMN2_intron7_ intronic-splicing-silencer (ISS-N1)mutation and correction of SMN2 aberrant splicing, by exploiting the non-homologous end-joining (NHEJ) pathway. Plasmids encoding Cas9-GFP under the control of CMV promoter, and selected gRNAs downstream to the Pol-III U6 promoter (Addgene) were transfected in HEK-293T cell line and in immortalized myoblasts derived from either healthy donors or SMA patients. Transfection efficiency was estimated as percentage of GFP-expressing cells (20-50% and 1-10%, respectively) and nuclease activity detected by Surveyor assay and target site sequencing. In particular, in SMA patient-derived myoblasts we detected mutations (indels) at the level of the induced DNA double-strand break at ~30% frequency. Levels of SMN restoration will be investigated by qPCR of the different species of SMN transcripts and by western blotting of SMN protein. The goal of this study is to provide an in vitro proof of principle of effective gene correction in SMA patient-derived cells. In the context of a multisystemic, complicated disease such as SMA, targeted genome editing strategy could represent an additional therapeutic tool