RESUMEN
Clinical success of autologous CD19-directed chimeric antigen receptor T cells (CAR Ts) in acute lymphoblastic leukemia and non-Hodgkin lymphoma suggests that CAR Ts may be a promising therapy for hematological malignancies, including multiple myeloma. However, autologous CAR T therapies have limitations that may impact clinical use, including lengthy vein-to-vein time and manufacturing constraints. Allogeneic CAR T (AlloCAR T) therapies may overcome these innate limitations of autologous CAR T therapies. Unlike autologous cell therapies, AlloCAR T therapies employ healthy donor T cells that are isolated in a manufacturing facility, engineered to express CARs with specificity for a tumor-associated antigen, and modified using gene-editing technology to limit T cell receptor (TCR)-mediated immune responses. Here, transcription activator-like effector nuclease (TALEN) gene editing of B cell maturation antigen (BCMA) CAR Ts was used to confer lymphodepletion resistance and reduced graft-versus-host disease (GvHD) potential. The safety profile of allogeneic BCMA CAR Ts was further enhanced by incorporating a CD20 mimotope-based intra-CAR off switch enabling effective CAR T elimination in the presence of rituximab. Allogeneic BCMA CAR Ts induced sustained antitumor responses in mice supplemented with human cytokines, and, most importantly, maintained their phenotype and potency after scale-up manufacturing. This novel off-the-shelf allogeneic BCMA CAR T product is a promising candidate for clinical evaluation.
Asunto(s)
Antígeno de Maduración de Linfocitos B/inmunología , Trasplante de Células/métodos , Inmunoterapia Adoptiva/métodos , Mieloma Múltiple/terapia , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Linfocitos T/trasplante , Animales , Antineoplásicos Inmunológicos/uso terapéutico , Antígeno de Maduración de Linfocitos B/genética , Donantes de Sangre , Línea Celular Tumoral , Trasplante de Células/efectos adversos , Citotoxicidad Inmunológica/genética , Edición Génica , Vectores Genéticos , Enfermedad Injerto contra Huésped/terapia , Humanos , Inmunoterapia Adoptiva/efectos adversos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mieloma Múltiple/patología , Supervivencia sin Progresión , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Rituximab/uso terapéutico , Linfocitos T/metabolismo , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética , Transducción Genética , Trasplante Homólogo/métodosRESUMEN
TALEN is one of the most widely used tools in the field of genome editing. It enables gene integration and gene inactivation in a highly efficient and specific fashion. Although very attractive, the apparent simplicity and high success rate of TALEN could be misleading for novices in the field of gene editing. Depending on the application, specific TALEN designs, activity assessments and screening strategies need to be adopted. Here we report different methods to efficiently perform TALEN-mediated gene integration and inactivation in different mammalian cell systems including induced pluripotent stem cells and delineate experimental examples associated with these approaches.
Asunto(s)
Marcación de Gen/métodos , Genoma/genética , Activación Transcripcional/genética , Transfección/métodos , Animales , Secuencia de Bases , Línea Celular , Proteínas de Unión al ADN/genética , Células HCT116 , Humanos , Datos de Secuencia MolecularRESUMEN
The ability to specifically engineer the genome of living cells at precise locations using rare-cutting designer endonucleases has broad implications for biotechnology and medicine, particularly for functional genomics, transgenics and gene therapy. However, the potential impact of chromosomal context and epigenetics on designer endonuclease-mediated genome editing is poorly understood. To address this question, we conducted a comprehensive analysis on the efficacy of 37 endonucleases derived from the quintessential I-CreI meganuclease that were specifically designed to cleave 39 different genomic targets. The analysis revealed that the efficiency of targeted mutagenesis at a given chromosomal locus is predictive of that of homologous gene targeting. Consequently, a strong genome-wide correlation was apparent between the efficiency of targeted mutagenesis (≤ 0.1% to ≈ 6%) with that of homologous gene targeting (≤ 0.1% to ≈ 15%). In contrast, the efficiency of targeted mutagenesis or homologous gene targeting at a given chromosomal locus does not correlate with the activity of individual endonucleases on transiently transfected substrates. Finally, we demonstrate that chromatin accessibility modulates the efficacy of rare-cutting endonucleases, accounting for strong position effects. Thus, chromosomal context and epigenetic mechanisms may play a major role in the efficiency rare-cutting endonuclease-induced genome engineering.
Asunto(s)
Efectos de la Posición Cromosómica , Enzimas de Restricción del ADN/metabolismo , Animales , Células CHO , Línea Celular , Cricetinae , Cricetulus , Enzimas de Restricción del ADN/química , Marcación de Gen , Ingeniería Genética , Genoma Humano , Humanos , MutagénesisRESUMEN
BACKGROUND: Autologous BCMA-specific CAR T-cell therapies have substantial activity in multiple myeloma (MM). However, due to logistical limitations and BCMAlow relapses, there is a need for alternatives. UCARTCS1 cells are 'off-the-shelf' allogeneic CAR T-cells derived from healthy donors targeting SLAMF7 (CS1), which is highly expressed in MM cells. In this study, we evaluated the preclinical activity of UCARTCS1 in MM cell lines, in bone marrow (BM) samples obtained from MM patients and in an MM mouse model. METHODS: Luciferase-transduced MM cell lines were incubated with UCARTCS1 cells or control (non-transduced, SLAMF7/TCRαß double knock-out) T-cells at different effector to target ratios for 24 hours. MM cell lysis was assessed by bioluminescence. Anti-MM activity of UCARTCS1 was also evaluated in 29 BM samples obtained from newly diagnosed patients (n=10), daratumumab-naïve relapsed/refractory patients (n=10) and daratumumab-refractory patients (n=9) in 24-hour flow cytometry-based cytotoxicity assays. Finally, UCARTCS1 activity was assessed in mouse xenograft models. RESULTS: UCARTCS1 cells induced potent CAR-mediated, and dose-dependent lysis of both MM cell lines and primary MM cells. There was no difference in ex vivo activity of UCARTCS1 between heavily pretreated and newly diagnosed patients. In addition, efficacy of UCARTCS1 was not affected by SLAMF7 expression level on MM cells, proportion of tumor cells, or frequency of regulatory T-cells in BM samples obtained from MM patients. UCARTCS1 treatment eliminated SLAMF7+ non-malignant immune cells in a dose-dependent manner, however lysis of normal cells was less pronounced compared to that of MM cells. Additionally, durable anti-MM responses were observed with UCARTCS1 in an MM xenograft model. CONCLUSIONS: These results demonstrate that UCARTCS1 has potent anti-MM activity against MM cell lines and primary MM cells, as well as in an MM xenograft model and support the evaluation of UCARTCS1 in patients with advanced MM.
Asunto(s)
Inmunoterapia Adoptiva , Mieloma Múltiple , Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Mieloma Múltiple/terapia , Mieloma Múltiple/inmunología , Mieloma Múltiple/patología , Humanos , Animales , Familia de Moléculas Señalizadoras de la Activación Linfocitaria/metabolismo , Ratones , Inmunoterapia Adoptiva/métodos , Receptores Quiméricos de Antígenos/metabolismo , Receptores Quiméricos de Antígenos/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto , Femenino , Línea Celular TumoralRESUMEN
Sickle cell disease is a devastating blood disorder that originates from a single point mutation in the HBB gene coding for hemoglobin. Here, we develop a GMP-compatible TALEN-mediated gene editing process enabling efficient HBB correction via a DNA repair template while minimizing risks associated with HBB inactivation. Comparing viral versus non-viral DNA repair template delivery in hematopoietic stem and progenitor cells in vitro, both strategies achieve comparable HBB correction and result in over 50% expression of normal adult hemoglobin in red blood cells without inducing ß-thalassemic phenotype. In an immunodeficient female mouse model, transplanted cells edited with the non-viral strategy exhibit higher engraftment and gene correction levels compared to those edited with the viral strategy. Transcriptomic analysis reveals that non-viral DNA repair template delivery mitigates P53-mediated toxicity and preserves high levels of long-term hematopoietic stem cells. This work paves the way for TALEN-based autologous gene therapy for sickle cell disease.
Asunto(s)
Anemia de Células Falciformes , Edición Génica , Terapia Genética , Células Madre Hematopoyéticas , Nucleasas de los Efectores Tipo Activadores de la Transcripción , Anemia de Células Falciformes/terapia , Anemia de Células Falciformes/genética , Edición Génica/métodos , Animales , Células Madre Hematopoyéticas/metabolismo , Humanos , Femenino , Ratones , Terapia Genética/métodos , Nucleasas de los Efectores Tipo Activadores de la Transcripción/metabolismo , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética , Trasplante de Células Madre Hematopoyéticas , Globinas beta/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Reparación del ADN , Mutación , Talasemia beta/terapia , Talasemia beta/genética , Modelos Animales de Enfermedad , Técnicas de Transferencia de GenRESUMEN
Integrative gene transfer is widely used for bioproduction, drug screening, and therapeutic applications but usual viral methods lead to random and multicopy insertions, contribute to unstable transgene expression and can disturb endogenous gene expression. Homologous targeting of an expression cassette using rare-cutting endonucleases is a potential solution; however the number of studied loci remains limited. Furthermore, the behavior and performance of various types of gene cassettes following gene targeting is poorly defined. Here we have evaluated three loci for gene targeting, including one locus compatible with the proposed Safe Harbor criteria for human translational applications. Using optimized conditions for homologous gene targeting, reporter genes under the control of different promoters were efficiently inserted at each locus in both sense and antisense orientations. Sustainable expression was achieved at all three loci without detectable disturbance of flanking gene expression. However, the promoter, the integration locus and the cassette orientation have a strong impact on transgene expression. Finally, single targeted integrations exhibited greatly improved transgene expression stability versus multicopy or random integration. Taken together, our data suggest a potential set of loci for site-specific transgene integration, suitable for a variety of biotechnological applications.
Asunto(s)
Expresión Génica , Marcación de Gen , Mutagénesis Insercional/métodos , Transgenes , Biotecnología/métodos , Línea Celular , Genes Reporteros , Inestabilidad Genómica , Humanos , Regiones Promotoras GenéticasRESUMEN
Despite the remarkable success of autologous chimeric antigen receptor (CAR) T cells, some patients relapse due to tumor antigen escape and low or uneven antigen expression, among other mechanisms. Therapeutic options after relapse are limited, emphasizing the need to optimize current approaches. In addition, there is a need to develop allogeneic "off-the-shelf" therapies from healthy donors that are readily available at the time of treatment decision and can overcome limitations of current autologous approaches. To address both challenges simultaneously, we generated a CD20xCD22 dual allogeneic CAR T cell. Herein, we demonstrate that allogeneic CD20x22 CAR T cells display robust, sustained and dose-dependent activity in vitro and in vivo, while efficiently targeting primary B-cell non-Hodgkin lymphoma (B-NHL) samples with heterogeneous levels of CD22 and CD20. Altogether, we provide preclinical proof-of-concept data for an allogeneic dual CAR T cell to overcome current mechanisms of resistance to CAR T-cell therapies in B-NHL, while providing a potential alternative to CD19 targeting.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Linfoma de Células B , Humanos , Receptores de Antígenos de Linfocitos T , Recurrencia Local de Neoplasia , Linfocitos T , Linfocitos B , Inmunoterapia Adoptiva , Antígenos CD19RESUMEN
Herpes simplex virus (HSV) infection is a leading cause of corneal blindness. However, keratoplasty is only rarely proposed due to the high frequency of graft failure and associated recurrences. Gene therapy of the corneal graft might provide sustained protection against HSV infection. To test that hypothesis, we designed a meganuclease specific to an HSV-1 DNA sequence coding for major capsid protein (UL19) and selected an adeno-associated virus type-2 as the vector. Meganuclease was transduced into corneas and its effect was challenged in vitro, ex vivo, and then in vivo in a rabbit HSV-1-infection model of stromal keratitis and endotheliitis. In vivo, meganuclease exposure resulted in fewer infected stromal and endothelial cells, and protected against corneal opacification and edema. Ex vivo, HSV-1 infection rates of meganuclease-treated human corneas were drastically reduced. Furthermore, genetically engineered corneas transplanted in vivo into rabbit eyes protected against HSV-1 infection. This genome-editing technology targeting HSV-1 opens new opportunities to manage severe post-herpetic corneal blindness by providing infected patients with genetically protected corneal transplants.
RESUMEN
Acute myeloid leukemia (AML) is a disease with high incidence of relapse that is originated and maintained from leukemia stem cells (LSCs). Hematopoietic stem cells can be distinguished from LSCs by an array of cell surface antigens such as CD123, thus a candidate to eliminate LSCs using a variety of approaches, including CAR T cells. Here, we evaluate the potential of allogeneic gene-edited CAR T cells targeting CD123 to eliminate LSCs (UCART123). UCART123 cells are TCRαßneg T cells generated from healthy donors using TALEN® gene-editing technology, decreasing the likelihood of graft vs host disease. As safety feature, cells express RQR8 to allow elimination with Rituximab. UCART123 effectively eliminates AML cells in vitro and in vivo with significant benefits in overall survival of AML-patient derived xenograft mice. Furthermore, UCART123 preferentially target AML over normal cells with modest toxicity to normal hematopoietic stem/progenitor cells. Together these results suggest that UCART123 represents an off-the shelf therapeutic approach for AML.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Leucemia Mieloide Aguda , Animales , Humanos , Subunidad alfa del Receptor de Interleucina-3/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/terapia , Ratones , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Linfocitos TRESUMEN
CD70 is highly expressed in renal cell carcinoma (RCC), with limited expression in normal tissue, making it an attractive CAR T target for an immunogenic solid tumor indication. Here we generated and characterized a panel of anti-CD70 single-chain fragment variable (scFv)-based CAR T cells. Despite the expression of CD70 on T cells, production of CAR T cells from a subset of scFvs with potent in vitro activity was achieved. Expression of CD70 CARs masked CD70 detection in cis and provided protection from CD70 CAR T cell-mediated fratricide. Two distinct classes of CAR T cells were identified with differing memory phenotype, activation status, and cytotoxic activity. Epitope mapping revealed that the two classes of CARs bind unique regions of CD70. CD70 CAR T cells displayed robust antitumor activity against RCC cell lines and patient-derived xenograft mouse models. Tissue cross-reactivity studies identified membrane staining in lymphocytes, thus matching the known expression pattern of CD70. In a cynomolgus monkey CD3-CD70 bispecific toxicity study, expected findings related to T-cell activation and elimination of CD70-expressing cells were observed, including cytokine release and loss of cellularity in lymphoid tissues. Finally, highly functional CD70 allogeneic CAR T cells were produced at large scale through elimination of the T-cell receptor by TALEN-based gene editing. Taken together, these efficacy and safety data support the evaluation of CD70 CAR T cells for the treatment of RCC and has led to the advancement of an allogeneic CD70 CAR T-cell candidate into phase I clinical trials. SIGNIFICANCE: These findings demonstrate the efficacy and safety of fratricide-resistant, allogeneic anti-CD70 CAR T cells targeting renal cell carcinoma and the impact of CAR epitope on functional activity. See related commentary by Adotévi and Galaine, p. 2517.
Asunto(s)
Carcinoma de Células Renales , Trasplante de Células Madre Hematopoyéticas , Neoplasias Renales , Animales , Ligando CD27 , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Humanos , Inmunoterapia Adoptiva , Neoplasias Renales/patología , Macaca fascicularis , Ratones , Linfocitos T/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Universal CAR T-cell therapies are poised to revolutionize cancer treatment and to improve patient outcomes. However, realizing these advantages in an allogeneic setting requires universal CAR T-cells that can kill target tumor cells, avoid depletion by the host immune system, and proliferate without attacking host tissues. Here, we describe the development of a novel immune-evasive universal CAR T-cells scaffold using precise TALEN-mediated gene editing and DNA matrices vectorized by recombinant adeno-associated virus 6. We simultaneously disrupt and repurpose the endogenous TRAC and B2M loci to generate TCRαß- and HLA-ABC-deficient T-cells expressing the CAR construct and the NK-inhibitor named HLA-E. This highly efficient gene editing process enables the engineered T-cells to evade NK cell and alloresponsive T-cell attacks and extend their persistence and antitumor activity in the presence of cytotoxic levels of NK cell in vivo and in vitro, respectively. This scaffold could enable the broad use of universal CAR T-cells in allogeneic settings and holds great promise for clinical applications.
Asunto(s)
Edición Génica , Nucleasas de los Efectores Tipo Activadores de la Transcripción , Humanos , Inmunoterapia Adoptiva , Receptores de Antígenos de Linfocitos T/genética , Linfocitos TRESUMEN
Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy with poor outcomes with conventional therapy. Nearly 100% of BPDCNs overexpress interleukin 3 receptor subunit alpha (CD123). Given that CD123 is differentially expressed on the surface of BPDCN cells, it has emerged as an attractive therapeutic target. UCART123 is an investigational product consisting of allogeneic T cells expressing an anti-CD123 chimeric antigen receptor (CAR), edited with TALEN® nucleases. In this study, we examine the antitumor activity of UCART123 in preclinical models of BPDCN. We report that UCART123 have selective antitumor activity against CD123-positive primary BPDCN samples (while sparing normal hematopoietic progenitor cells) in the in vitro cytotoxicity and T cell degranulation assays; supported by the increased secretion of IFNγ by UCART123 cells when cultured in the presence of BPDCN cells. UCART123 eradicate BPDCN and result in long-term disease-free survival in a subset of primary patient-derived BPDCN xenograft mouse models. One potential challenge of CD123 targeting therapies is the loss of CD123 antigen through diverse genetic mechanisms, an event observed in one of three BPDCN PDX studied. In summary, these results provide a preclinical proof-of-principle that allogeneic UCART123 cells have potent anti-BPDCN activity.
Asunto(s)
Neoplasias Hematológicas , Trasplante de Células Madre Hematopoyéticas , Trastornos Mieloproliferativos , Neoplasias Cutáneas , Enfermedad Aguda , Animales , Células Dendríticas/metabolismo , Neoplasias Hematológicas/tratamiento farmacológico , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Subunidad alfa del Receptor de Interleucina-3/metabolismo , Ratones , Trastornos Mieloproliferativos/metabolismo , Neoplasias Cutáneas/patologíaRESUMEN
The ability of pathogens to escape the host's immune response is crucial for the establishment of persistent infections and can influence virulence. Recombination has been observed to contribute to this process by generating novel genetic variants. Although distinctive recombination patterns have been described in many viral pathogens, little is known about the influence of biases in the recombination process itself relative to selective forces acting on newly formed recombinants. Understanding these influences is important for determining how recombination contributes to pathogen genome and proteome evolution. Most previous research on recombination-driven protein evolution has focused on relatively simple proteins, usually in the context of directed evolution experiments. Here, we study recombination in the envelope gene of HIV-1 between primary isolates belonging to subtypes that recombine naturally in the HIV/AIDS pandemic. By characterizing the early steps in the generation of recombinants, we provide novel insights into the evolutionary forces that shape recombination patterns within viral populations. Specifically, we show that the combined effects of mechanistic processes that determine the locations of recombination breakpoints across the HIV-1 envelope gene, and purifying selection acting against dysfunctional recombinants, can explain almost the entire distribution of breakpoints found within this gene in nature. These constraints account for the surprising paucity of recombination breakpoints found in infected individuals within this highly variable gene. Thus, the apparent randomness of HIV evolution via recombination may in fact be relatively more predictable than anticipated. In addition, the dominance of purifying selection in localized areas of the HIV genome defines regions where functional constraints on recombinants appear particularly strong, pointing to vulnerable aspects of HIV biology.
Asunto(s)
VIH-1/genética , Recombinación Genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Secuencia de Bases , Infecciones por VIH/genética , Humanos , Reacción en Cadena de la Polimerasa , ARN Viral/genéticaRESUMEN
We evaluate gene editing of HSV in a well-established mouse model, using adeno-associated virus (AAV)-delivered meganucleases, as a potentially curative approach to treat latent HSV infection. Here we show that AAV-delivered meganucleases, but not CRISPR/Cas9, mediate highly efficient gene editing of HSV, eliminating over 90% of latent virus from superior cervical ganglia. Single-cell RNA sequencing demonstrates that both HSV and individual AAV serotypes are non-randomly distributed among neuronal subsets in ganglia, implying that improved delivery to all neuronal subsets may lead to even more complete elimination of HSV. As predicted, delivery of meganucleases using a triple AAV serotype combination results in the greatest decrease in ganglionic HSV loads. The levels of HSV elimination observed in these studies, if translated to humans, would likely significantly reduce HSV reactivation, shedding, and lesions. Further optimization of meganuclease delivery and activity is likely possible, and may offer a pathway to a cure for HSV infection.
Asunto(s)
Desoxirribonucleasas/genética , Dependovirus/genética , Infecciones del Ojo/terapia , Edición Génica/métodos , Herpes Simple/terapia , Herpesvirus Humano 1/genética , Latencia del Virus/genética , Animales , Sistemas CRISPR-Cas/genética , Células Cultivadas , Chlorocebus aethiops , Infecciones del Ojo/genética , Infecciones del Ojo/virología , Femenino , Células HEK293 , Herpes Simple/genética , Herpesvirus Humano 1/patogenicidad , Humanos , Ratones , Neuronas/metabolismo , Neuronas/virología , RNA-Seq , Análisis de la Célula Individual , Ganglio Cervical Superior/metabolismo , Ganglio Cervical Superior/virología , Células VeroRESUMEN
The human immunodeficiency virus (HIV) population is characterised by extensive genetic variability that results from high error and recombination rates of the reverse transcription process, and from the fast turnover of virions in HIV-infected individuals. Among the viral variants encountered at the global scale, recombinant forms are extremely abundant. Some of these recombinants (known as circulating recombinant forms) become fixed and undergo rapid expansion in the population. The reasons underlying their epidemiological success remain at present poorly understood and constitute a fascinating area for future research to improve our understanding of immune escape, pathogenicity and transmission. Recombinant viruses are generated during reverse transcription as a consequence of template switching between the two genetically different genomic RNAs present in a heterozygous virus. Recombination can thereby generate shortcuts in evolution by producing mosaic reverse transcription products of parental genomes. Therefore, in a single infectious cycle multiple mutations that are positively selected can be combined or, conversely, negatively selected mutations can be removed. Recombination is therefore involved in different aspects of HIV evolution, adaptation to its host, and escape from antiviral treatments.
Asunto(s)
Variación Genética , VIH/genética , Recombinación Genética , Genoma Viral , VIH/clasificación , VIH/inmunología , VIH/aislamiento & purificación , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Infecciones por VIH/transmisión , Infecciones por VIH/virología , HumanosRESUMEN
Retroviral recombination results from strand switching, during reverse transcription, between the two copies of genomic RNA present in the virus. We analysed recombination in part of the envelope gene, between HIV-1 subtype A and D strains. After a single infection cycle, breakpoints clustered in regions corresponding to the constant portions of Env. With some exceptions, a similar distribution was observed after multiple infection cycles, and among recombinant sequences in the HIV Sequence Database. We compared the experimental data with computer simulations made using a program that only allows recombination to occur whenever an identical base is present in the aligned parental RNAs. Experimental recombination was more frequent than expected on the basis of simulated recombination when, in a region spanning 40 nt from the 5' border of a breakpoint, no more than two discordant bases between the parental RNAs were present. When these requirements were not fulfilled, breakpoints were distributed randomly along the RNA, closer to the distribution predicted by computer simulation. A significant preference for recombination was also observed for regions containing homopolymeric stretches. These results define, for the first time, local sequence determinants for recombination between divergent HIV-1 isolates.
Asunto(s)
ADN Viral/química , VIH-1/genética , Recombinación Genética , Secuencia de Bases , Línea Celular , Mapeo Cromosómico , Simulación por Computador , Roturas del ADN , ADN Recombinante/química , Proteína gp120 de Envoltorio del VIH/genética , VIH-1/clasificación , VIH-1/fisiología , Humanos , Datos de Secuencia Molecular , ARN Viral/química , Alineación de Secuencia , Análisis de Secuencia de ADN , Replicación ViralRESUMEN
Retroviral recombination has been suggested as a useful way to modify retroviral vectors. The possibility to combine two multiply deleted retroviral vectors into a novel vector was evaluated. To investigate this possibility we have constructed two defective vectors containing a shared internal ribosome entry site (IRES). The IRES was selected for its complex secondary structure, a feature described to favour retroviral recombination. The IRES was expected to promote a recombination event leading to the formation of a unique, functional retroviral vector. By supporting expression of two transgenes from a single promoter, this sequence was also expected to allow straightforward detection of the recombination event. The present data confirms the achievement of recombination-dependent rescue, albeit at low efficiency. Unexpectedly, a preferential use of the packaging signal (Psi) for recombination was observed, as compared to the IRES. Together these observations mitigate the idea of using this technique for the design of retroviral vectors.
Asunto(s)
Vectores Genéticos , Recombinación Genética , Retroviridae/genética , Línea Celular , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Transcripción Reversa , Ribosomas/metabolismo , Transducción Genética , Transfección , Transgenes/genética , Transgenes/efectos de la radiación , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
BACKGROUND: HIV-1 recombination between different subtypes has a major impact on the global epidemic. The generation of these intersubtype recombinants follows a defined set of events starting with dual infection of a host cell, heterodiploid virus production, strand transfers during reverse transcription, and then selection. In this study, recombination frequencies were measured in the C1-C4 regions of the envelope gene in the presence (using a multiple cycle infection system) and absence (in vitro reverse transcription and single cycle infection systems) of selection for replication-competent virus. Ugandan subtypes A and D HIV-1 env sequences (115-A, 120-A, 89-D, 122-D, 126-D) were employed in all three assay systems. These subtypes co-circulate in East Africa and frequently recombine in this human population. RESULTS: Increased sequence identity between viruses or RNA templates resulted in increased recombination frequencies, with the exception of the 115-A virus or RNA template. Analyses of the recombination breakpoints and mechanistic studies revealed that the presence of a recombination hotspot in the C3/V4 env region, unique to 115-A as donor RNA, could account for the higher recombination frequencies with the 115-A virus/template. Single-cycle infections supported proportionally less recombination than the in vitro reverse transcription assay but both systems still had significantly higher recombination frequencies than observed in the multiple-cycle virus replication system. In the multiple cycle assay, increased replicative fitness of one HIV-1 over the other in a dual infection dramatically decreased recombination frequencies. CONCLUSION: Sequence variation at specific sites between HIV-1 isolates can introduce unique recombination hotspots, which increase recombination frequencies and skew the general observation that decreased HIV-1 sequence identity reduces recombination rates. These findings also suggest that the majority of intra- or intersubtype A/D HIV-1 recombinants, generated with each round of infection, are not replication-competent and do not survive in the multiple-cycle system. Ability of one HIV-1 isolate to outgrow the other leads to reduced co-infections, heterozygous virus production, and recombination frequencies.
Asunto(s)
VIH-1/genética , Recombinación Genética , Secuencia de Bases , Línea Celular , Mapeo Cromosómico , Genes env , VIH-1/aislamiento & purificación , VIH-1/fisiología , Humanos , ARN Viral/química , Uganda , Replicación ViralRESUMEN
The importance of recombination in retroviral evolution has been acknowledged for several decades. Consequently, after the identification of HIV as the etiological agent of AIDS, it was suspected that recombination could also play a central role in the evolution of this virus. However, only recently, extensive epidemiologic studies of HIV infections worldwide have provided an estimate for the occurrence of recombination in vivo, unveiling recombination frequencies that dwarf those initially expected. Nowadays, recombination is regarded as an integral part of the infectious cycle of this retrovirus, which impacts on diagnosis and treatment of infections, especially when genetically distant viruses have been at the origin of the recombinant forms. Retroviral recombination is observed when two genetically divergent genomic RNA molecules are present in the same viral particle, and arises during the reverse transcription step. This review focuses on the mechanisms that have been proposed to account for the occurrence of recombination in retroviruses, from the strand displacement model, according to which recombination occurs during second DNA strand synthesis; to the description of the factors responsible for copy-choice recombination during first DNA strand synthesis, such as the presence of breaks, pause sites, or secondary structures in the genomic RNA. Most of these models have been supported by experimental data obtained from in vitro reconstituted systems or from cell infection studies using academic model sequences. The situation in vivo is expected to be more complex, since several factors come into play when recombination involves relatively distant isolates, as in the case of inter-subtype recombination. At present, it is clear that further studies are needed in order to evaluate whether a prevailing mechanism exists for in vivo recombination, and these studies will also be essential for understanding how the underlying mechanisms of recombination contribute to the evolution of HIV.
Asunto(s)
VIH-1/genética , Virus Reordenados , Recombinación Genética , Evolución Molecular , Infecciones por VIH/virología , HumanosRESUMEN
We report the isolation of the mouse JNK/SAPKalpha gene, the determination of its exon/intron organization and the characterization of its promoter region. The mouse JNK/SAPKalpha gene spans a region of 36 kbp and contains 13 exons, which represent about 8% of the gene sequence. Major JNK/SAPKalpha splice variants (I and II) are generated by alternative splicing of exons 7 and 8, respectively, whereas minor variants (III and IV) are generated using cryptic sites located inside exon 9. The regulatory elements of the JNK/SAPKalpha gene are located in a 400-bp region placed upstream of the first exon. The gene lacks a TATA element and the initiation of transcription is located inside a 1-kbp CG island. Two regulatory regions located at -98/-69 and -69/-30 were defined by deletion analysis of the promoter.