RESUMO
Application of viral vectors in gene delivery is attracting widespread attention but is hampered by the absence of control over transduction, which may lead to non-selective transduction with adverse side effects. To overcome some of these limitations, we proposed an unnatural amino acid aided caging-uncaging strategy for controlling the transduction capability of a viral vector. In this proof-of-principle study, we first expanded the genetic code of the lentiviral vector to incorporate an azido-containing unnatural amino acid (Nϵ-2-azidoethyloxycarbonyl-l-lysine, NAEK) site specifically within a lentiviral envelope protein. Screening of the resultant vectors indicated that NAEK incorporation at Y77 and Y116 was capable of inactivating viral transduction upon click conjugation with a photo-cleavable chemical molecule (T1). Exposure of the chimeric viral vector (Y77-T1) to UVA light subsequently removed the photo-caging group and restored the transduction capability of lentiviral vector both in vitro and in vivo. Our results indicate that the use of the photo-uncage activation procedure can reverse deactivated lentiviral vectors and thus enable regulation of viral transduction in a switchable manner. The methods presented here may be a general approach for generating various switchable vectors that respond to different stimulations and adapt to different viral vectors.
Assuntos
Vetores Genéticos/genética , Lentivirus/genética , Lisina/análogos & derivados , Transdução Genética , Azidas/efeitos da radiação , Linhagem Celular , Terapia Genética/métodos , Vetores Genéticos/efeitos da radiação , HIV-1/genética , Humanos , Lentivirus/efeitos da radiação , Lisina/genética , Lisina/efeitos da radiação , Raios Ultravioleta , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/efeitos da radiaçãoRESUMO
Disentangling cellular heterogeneity is a challenge in many fields, particularly in the stem cell and cancer biology fields. Here we demonstrate how to combine viral genetic barcoding with high-throughput sequencing to track single cells in a heterogeneous population. We use this technique to track the in vivo differentiation of unitary hematopoietic stem cells (HSCs). The results are consistent with single-cell transplantation studies but require two orders of magnitude fewer mice. In addition to its high throughput, the high sensitivity of the technique allows for a direct examination of the clonality of sparse cell populations such as HSCs. We show how these capabilities offer a clonal perspective of the HSC differentiation process. In particular, our data suggest that HSCs do not equally contribute to blood cells after irradiation-mediated transplantation, and that two distinct HSC differentiation patterns co-exist in the same recipient mouse after irradiation. This technique can be applied to any virus-accessible cell type for both in vitro and in vivo processes.
Assuntos
Código de Barras de DNA Taxonômico/métodos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/virologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Lentivirus/genética , Análise de Célula Única/métodos , Animais , Diferenciação Celular/genética , Diferenciação Celular/efeitos da radiação , Linhagem da Célula/genética , Linhagem da Célula/efeitos da radiação , Células Clonais , Biblioteca Gênica , Células-Tronco Hematopoéticas/efeitos da radiação , Lentivirus/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Radiação , Análise de Sequência de DNARESUMO
Inactivated viruses are important tools for vaccine development and gene transfer. 8-Methoxypsoralen (8-MOP) and long-wavelength ultraviolet irradiation (LWUVI) inactivates many viruses. Toxicity limits its use in animals and humans. Toxicological and photosensitizing properties of riboflavin make it suitable for virus inactivation in preparations for biological use. Viruses expressing beta-galactosidase were mixed with either 8-MOP (1.5mM) or riboflavin (50 microM) and exposed to LWUVI (365 nm) for 2 h. Virus activity was determined by limiting dilution. The half-life of the adenovirus preparation treated with 8-MOP was 8.28 ns(-1) and 36.5 ns(-1) after treatment with riboflavin. Despite the difference in half-life, both preparations were completely inactivated within 45 min. In contrast, the half-lives for adeno-associated virus (AAV) preparations were similar (63 ns(-1) 8-MOP vs. 67 ns(-1) riboflavin). Each AAV preparation was fully inactivated within 90 min. The half-life of lentivirus was 193.4 ns(-1) after treatment with 8-MOP and 208 ns(-1) after exposure to riboflavin. Virus treated with riboflavin was inactivated within 20 min. Virus exposed to 8-MOP was inactivated in 90 min. DNA and RNA viruses can be inactivated by riboflavin and LWUVI and used in physiological systems sensitive to other photochemicals.
