RESUMO
Receptor targeting of vector particles is a key technology to enable cell type-specific in vivo gene delivery. For example, T cells in humanized mouse models can be modified by lentiviral vectors (LVs) targeted to human T-cell markers to enable them to express chimeric antigen receptors (CARs). Here, we provide detailed protocols for the generation of CD4- and CD8-targeted LVs (which takes ~9 d in total). We also describe how to humanize immunodeficient mice with hematopoietic stem cells (which takes 12-16 weeks) and precondition (over 5 d) and administer the vector stocks. Conversion of the targeted cell type is monitored by PCR and flow cytometry of blood samples. A few weeks after administration, ~10% of the targeted T-cell subtype can be expected to have converted to CAR T cells. By closely following the protocol, sufficient vector stock for the genetic manipulation of 10-15 humanized mice is obtained. We also discuss how the protocol can be easily adapted to use LVs targeted to other types of receptors and/or for delivery of other genes of interest.
Assuntos
Engenharia Genética/métodos , Linfócitos T/metabolismo , Animais , Antígenos CD/metabolismo , Células HEK293 , Humanos , Lentivirus/genética , Camundongos , Modelos Animais , Receptores de Antígenos Quiméricos/genéticaRESUMO
Adoptive T cell immunotherapy in combination with gene therapy is a promising treatment concept for chronic infections and cancer. Recently, receptor-targeted lentiviral vectors (LVs) were shown to enable selective gene transfer into particular types of lymphocytes both in vitro and in vivo. This approach might facilitate the genetic engineering of a patient's own T lymphocytes, possibly even shifting this concept from personalized medicine to an off-the shelf therapy in future. Here, we describe novel high-affinity binders for CD8 consisting of designed ankyrin repeat proteins (DARPins), which were selected to bind to the CD8 receptor of human and nonhuman primate (NHP) cells. These binders were identified by ribosome display screening of DARPin libraries using recombinant human CD8 followed by receptor binding analysis on primary lymphocytes. CD8-targeted LVs (CD8-LVs) were then generated that delivered genes exclusively and specifically to human and NHP T lymphocytes by using the same targeting domain. These CD8-LVs were as specific for human T lymphocytes as their single-chain variable fragment-based counterpart, but they could be produced to higher titers. Moreover, they were superior in transducing cytotoxic T cells both in vitro and in vivo when equal particle numbers were applied. Since the here described CD8-LVs transduced primary T lymphocytes from NHP and human donors equally well, they offer the opportunity for preclinical studies in different animal models including large animals such as NHPs without the need for modifications in vector design.
Assuntos
Repetição de Anquirina , Linfócitos T CD8-Positivos/metabolismo , Vetores Genéticos , Receptores de Antígenos de Linfócitos T/metabolismo , Anticorpos de Cadeia Única/genética , Animais , Linhagem Celular , Doença Crônica/terapia , Técnicas de Transferência de Genes , Terapia Genética/métodos , Células HEK293 , Humanos , Lentivirus , Leucócitos Mononucleares , Macaca mulatta , Macaca nemestrina , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes de Fusão/genética , Linfócitos T Citotóxicos/metabolismo , Transdução GenéticaRESUMO
Chimeric antigen receptor (CAR) T cells are in prime focus of current research in cancer immunotherapy. Facilitating CAR T cell generation is among the top goals. We have recently demonstrated direct in vivo generation of human CD19-CAR T cells by targeting CD8+ cells using lentiviral vectors (LVs). The anti-tumor potency of in vivo generated CAR T cells was assessed in human PBMC-transplanted NSG mice carrying i.v. injected CD19+ Nalm-6 tumor cells. A single injection of CD8-targeted LV delivering CD19-CAR was sufficient to completely eliminate the tumor cells from bone marrow and spleen, whereas control animals contained high levels of CD19+ cells. Tumor elimination was due to in vivo generated CAR+ cells. Notably, these were not only composed of T lymphocytes but also included CAR+ natural killer cells (NK and NKT). This is the first demonstration of tumor elimination by in vivo generated human CAR T cells.
RESUMO
BACKGROUND: Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes high fever, rash, and recurrent arthritis in humans. It has efficiently adapted to Aedes albopictus, which also inhabits temperate regions and currently causes large outbreaks in the Caribbean and Latin America. Ebola virus (EBOV) is a member of the filovirus family. It causes the Ebola virus disease (EDV), formerly known as Ebola hemorrhagic fever in humans and has a mortality rate of up to 70 %. The last outbreak in Western Africa was the largest in history and has caused approximately 25,000 cases and 10,000 deaths. For both viral infections no specific treatment or licensed vaccine is currently available. The bis-hexasulfonated naphthylurea, suramin, is used as a treatment for trypanosome-caused African river blindness. As a competitive inhibitor of heparin, suramin has been described to have anti-viral activity. METHODS: We tested the activity of suramin during CHIKV or Ebola virus infection, using CHIKV and Ebola envelope glycoprotein pseudotyped lentiviral vectors and wild-type CHIKV and Ebola virus. RESULTS: Suramin efficiently inhibited CHIKV and Ebola envelope-mediated gene transfer while vesicular stomatitis virus G protein pseudotyped vectors were only marginally affected. In addition, suramin was able to inhibit wild-type CHIKV and Ebola virus replication in vitro. Inhibition occurred at early time points during CHIKV infection. CONCLUSION: Suramin, also known as Germanin or Bayer-205, is a market-authorized drug, however shows significant side effects, which probably prevents its use as a CHIKV drug, but due to the high lethality of Ebola virus infections, suramin might be valuable against Ebola infections.
Assuntos
Antivirais/farmacologia , Febre de Chikungunya/virologia , Vírus Chikungunya/efeitos dos fármacos , Ebolavirus/efeitos dos fármacos , Doença pelo Vírus Ebola/virologia , Suramina/farmacologia , Internalização do Vírus/efeitos dos fármacos , Animais , Linhagem Celular , Vírus Chikungunya/genética , Vírus Chikungunya/fisiologia , Ebolavirus/genética , Ebolavirus/fisiologia , Humanos , Replicação Viral/efeitos dos fármacosRESUMO
Myxobacteria produce secondary metabolites many of which were described to have various biological effects including anti-fungal, anti-bacterial and anti-viral activity. The majority of these metabolites are novel scaffolds with unique modes-of-action and hence might be potential leads for drug discovery. Here, we tested a myxobacterial natural product library for compounds with inhibitory activity against Ebola virus (EBOV). The assay was performed with a surrogate system using Ebola envelope glycoprotein (GP) pseudotyped lentiviral vectors. EBOV specificity was proven by counter-screening with vesicular stomatitis virus G protein pseudotyped vectors. Two compounds were identified that preferentially inhibited EBOV GP mediated cell entry: Chondramides that act on the actin skeleton but might be too toxic and noricumazole A, a potassium channel inhibitor, which might constitute a novel pathway to inhibit Ebola virus cell entry.
Assuntos
Antivirais/farmacologia , Produtos Biológicos/farmacologia , Descoberta de Drogas , Ebolavirus/efeitos dos fármacos , Ebolavirus/fisiologia , Bibliotecas de Moléculas Pequenas , Internalização do Vírus/efeitos dos fármacos , Actinas/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Engenharia Genética , Vetores Genéticos/genética , Doença pelo Vírus Ebola/tratamento farmacológico , Doença pelo Vírus Ebola/virologia , Humanos , Myxococcales/química , Myxococcales/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes chikungunya fever and has infected millions of people mainly in developing countries. The associated disease is characterized by rash, high fever, and severe arthritis that can persist for years. CHIKV has adapted to Aedes albopictus, which also inhabits temperate regions including Europe and the United States of America. CHIKV has recently caused large outbreaks in Latin America. No treatment or licensed CHIKV vaccine exists. Traditional medicines are known to have anti-viral effects; therefore, we examined whether curcumin or Boswellia serrata gum resin extract have antiviral activity against CHIKV. Both compounds blocked entry of CHIKV Env-pseudotyped lentiviral vectors and inhibited CHIKV infection in vitro. In addition, vesicular stomatitis virus vector particles and viral infections were also inhibited to the same extent, indicating a broad antiviral activity. Although the bioavailability of these compounds is rather poor, they might be used as a lead structure to develop more effective antiviral drugs or might be used topically to prevent CHIKV spread in the skin after mosquito bites.