A comparative evaluation of gene transfer into blood cells using the same retroviral backbone for independent expression of the EGFP and deltaLNGFR marker genes.

BACKGROUND AND OBJECTIVES: Retroviral vectors are widely used to deliver foreign genes to hematopoietic stem cells (HSC). Improvement of marking protocols needs reporter genes to allow rapid detection and efficient selection of transduced cells. The great potential of EGFP and LNGFR as reporter systems prompted us to compare them simultaneously, using the same retroviral backbone and the same gene transfer procedures. DESIGN AND METHODS: The EGFP and LNGFR coding sequences were separately cloned into the MFG retroviral backbone. A cloning strategy assuring that both genes utilize the same ATG as the start codon was adopted. Marker gene expression, viral titers, transduction efficiency, and vector stability were evaluated in expanded amphotropic packaging clones and human hematopoietic cell lines by flow cytometry and PCR analysis. Vectors were also tested for their ability to transduce CD34+ peripheral blood cells. RESULTS: A significantly larger number of MFG- LNGFR packaging clones were obtained that produced high viral titers. A direct correlation between viral titer and marker gene expression in packaging clones was demonstrated for both constructs. Similar expression kinetics and absence of in vitro toxicity in transduced cells were also observed for both constructs. Successful infection of CD34+ cells was achieved even after a short time of exposure to recombinant viruses. INTERPRETATION AND CONCLUSIONS: Our results demonstrate that EGFP and LNGFR marker genes are equally useful for a rapid, specific and non-toxic detection of transduced cells. The MFG-EGFP construct appears useful to optimize gene transfer protocols in vitro. On the other hand, the MFG-LNGFR construct, for making possible a more efficient selection of high titer producer clones, as well as for safety and adaptability to the in vivo use, is more suitable for clinical applications.

Herpes simplex virus chronically infected human T lymphocytes are susceptible to HIV-1 superinfection and support HIV-1 pseudotyping.

Peripheral blood lymphocytes (PBL) and CEM CD4+ T-cell line can be infected by herpes simplex virus-1 (HSV-1). CEM cells were characterized as a cellular model to study interactions occurring between HSV-1 and HIV-1. Virtually all cells were persistently infected by HSV-1 (CEM(HSV)) and expressed the latency associated transcripts, whereas only a fraction tested positive for HSV-antigens. CD4 and CXCR-4 expression and function were not affected in CEM(HSV) cells and no significant increase of deoxyribonucleotide pools was noticed. Superinfection of CEM(HSV) cells with HIV-1 led to a cell line chronically infected by both viruses (CEM(HSV/HSV)). Evidence was also obtained that this cell line can produce HIV-1 pseudotyped by HSV-1 envelope. These results may have important implications for a better understanding of AIDS pathogenesis.

Inducible expression of herpes simplex virus thymidine kinase from a bicistronic HIV1 vector.

The possibility of protecting human CD4+ lymphocytes from human immunodeficiency virus type 1 (HIV1) infection, through a suicide mechanism elicited by the HIV1 transcription apparatus itself, offers a potentially useful approach for gene therapy of the acquired immunodeficiency syndrome. A replication-defective lentiviral HIV1 vector (HYIRES-TK) was designed to carry both the hygromycin (Hy) phosphotransferase gene for positive selection and the thymidine kinase (TK) gene of herpes simplex virus driven by the viral long terminal repeat (LTR). The internal ribosome entry site (IRES) from encephalomyocarditis virus was placed between the two genes for their efficient simultaneous translation. Transient expression of active TK into transfected COS-1 cells was shown to be induced by Tat and Rev over a detectable basal level. By providing the missing viral proteins in trans, recombinant viruses were generated and used to transduce Jurkat cells. The Hy-resistant population of cells was sensitive to ganciclovir (GCV) and acyclovir (ACV), a result consistent with a basal level of TK expression. Cocultivation of transduced cells with cells chronically infected with HIV in the presence of 10 microM ACV, a concentration non-toxic for the uninfected cells, resulted in increased killing of cells transduced with the HY-IRES-TK vector. These data indicate that two genes can be expressed from the viral LTR in the context of an HIV1 vector, with the aid of an IRES sequence. The expression is inducible by the HIV proteins Tat and Rev and it is possible to specifically kill infected cells with subtoxic concentrations of drug. To decrease the sensitivity of the transduced cells towards GCV, a variant vector expressing a truncated TK was constructed. The truncated version was expressed at levels similar to those of wild-type TK but induced sensitivity towards GCV in transduced cells that was intermediate between that of untransduced cells and of cells expressing wild-type TK.

[Two cases of amniotic band disruption complex (author's transl)]. / Due casi di complesso malformativo e deformativo da briglie amniotiche.

Two cases of amniotic band disruption complex are described: in both newborns severe craniofacial defects are associated with limb defects. The quality of survival may be satisfactory when surgical correction of limbs and craniofacial defects is possible, like in the newborn less severely affected. Recurrence risk is very low, with respect to the sporadic nature of this disorder. A more accurate diagnosis may be done through placenta and fetal sac studies.