Biomedical subjects

David J Looney

Publications and source records attributed to David J Looney.

8 recordsLinked to original sources

PubMed2005

Inhibition of HIV-1 replication by siRNA targeting conserved regions of gag/pol.

Short interfering RNAs (siRNAs) targeting HIV-1gag, vif, tat, rev and host CD4 and CCR5 have been reported to inhibit HIV replication. However, the sequence divergence of HIV and the concentration dependence of siRNA activity represent significant challenges to RNAi mediated inhibition. To determine the parameters of RNAi in suppression of HIV-1 we screened seven siRNA candidates targeting highly conserved regions of gag/pol, based on target site GC content, for antiviral activity at varying concentrations. Only two of these inhibited CA-p24 production more than 50%, 2064 and 2161. Activity varied with concentration, with 100 nM producing optimal suppression. Requirements for target sequence conservation and activity over a range of concentrations may severely limit the number of siRNA candidates for therapeutic development.

Cell Line↗

PubMed2005

Characterization of human immunodeficiency virus (HIV)-2 vector mobilization by HIV-1.

Conditionally replicating human immunodeficiency virus type 2 (crHIV-2) vectors can compete with HIV-1 for packaging in HIV-1-infected cells, indicating that the mobilization of vectors could selectively target as well as protect reservoirs susceptible to HIV-1 infection. The incorporation of HIV-1-specific antiviral transgenes in crHIV-2 vectors, although increasing the direct antiviral effect, may decrease mobilization and transmission to surrounding cells. To investigate how HIV-1-specific catalytic RNA cassettes (ribozymes) affect this balance between antiviral activity and mobilization, crHIV-2 vectors shown to display anti-HIV-1 activity were packaged by HIV-2 and used to transduce cells previously infected with HIV-1 or to transduce uninfected cells that were subsequently challenged with HIV-1. Vector mobilization was greater when HIV-1-infected cells were transduced with vector than when transduced cells were infected with HIV-1, and approximately 3-fold lower vector production was observed in cultures transduced with vectors expressing anti-HIV-1 ribozymes. Vector and antiviral effects could be transferred to new cultures by passaging supernatants to fresh cultures. No evidence of recombination with HIV-1 was observed. Vector mobilization and protection from HIV-1 infection were also demonstrated in human peripheral blood mononuclear cells. These data suggest that strategies employing vector mobilization for HIV-1 gene therapy should use vectors with maximal antiviral potency, despite resulting reductions in mobilization of the vector.

Base Sequence↗

PubMed2004

Small interfering RNA-induced transcriptional gene silencing in human cells.

Small interfering RNA (siRNA) and microRNA silence genes at the transcriptional, posttranscriptional, and/or translational level. Using human tissue culture cells, we show that promoter-directed siRNA inhibits transcription of an integrated, proviral, elongation factor 1alpha (EF1A) promoter-green fluorescent protein reporter gene and of endogenous EF1A. Silencing was associated with DNA methylation of the targeted sequence, and it required either active transport of siRNA into the nucleus or permeabilization of the nuclear envelope by lentiviral transduction. These results demonstrate that siRNA-directed transcriptional silencing is conserved in mammals, providing a means to inhibit mammalian gene function.

Active Transport, Cell Nucleus↗

PubMed2004

Feline immunodeficiency virus: a concise review.

Among non-primate vertebrates, feline immunodeficiency virus (FIV) infection in the cat may be the closest model of human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS). Clinical evolution and immunological and virological relationships between human HIV/AIDS and disease produced by FIV infection in cats are very close. These similarities should facilitate progress in the understanding of mechanisms of viral infection and immunopathology, and make this model potentially very valuable in evaluation of experimental therapeutic approaches to AIDS in man. Development of feline immunodeficiency virus vectors bearing therapeutic genes targeting different human diseases is a promising strategy for gene therapy, despite some recent studies which suggest that despite lack of evidence of infection of man by FIV, additional epidemiological surveillance may be indicated to determine if transmission can occur from this close companion to humans in some circumstances.

Animals↗

PubMed2004

Transduction of cell lines and primary cells by FIV-packaged HIV vectors.

Human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus, and feline immunodeficiency virus (FIV) are capable of packaging viral RNA derived from heterologous as well as homologous lentiviruses, a phenomenon referred to as "cross packaging." To remove the possibility of seroconversion to HIV proteins, and to avoid potential problems arising due to targeting of vector or packaging construct by antiviral genes, we investigated the feasibility of using an FIV-based packaging system to deliver human immunodeficiency virus type 2 (HIV-2)-based vectors bearing anti-HIV-1 RNA expression cassettes to target cells. In the absence of FIV rev, FIV was packaged by HIV-2 at only 3% the efficiency of FIV packaging by FIV, but this was increased to 39% of homologous controls by supplying FIV rev in trans. HIV-2 vectors were packaged by FIV at levels equal to or exceeding the homologous HIV-2 packaging system in the absence of HIV-1 tat and rev, and levels increased approximately four- to fivefold with the addition of tat and rev in trans. HIV-2 vectors bearing a polyribozyme cassette targeting multiple regions of HIV RNA were efficiently packaged by FIV and transferred to target cells. Upon challenge with cell-free HIV-1 (m.o.i. = 0.1) a significant reduction in replication was observed. These findings demonstrate that packaging HIV vectors with FIV is a viable alternative, which avoids use of HIV structural proteins.

Cell Line↗

PubMed2004

Characterization of a mobilization-competent simian immunodeficiency virus (SIV) vector containing a ribozyme against SIV polymerase.

Exploitation of the intracellular virus machinery within infected cells to drive an anti-viral gene therapy vector may prove to be a feasible alternative to reducing viral loads or overall virus infectivity while propagating the spread of a therapeutic vector. Using a simian immunodeficiency virus (SIV)-based system, it was shown that the pre-existing retroviral biological machinery within SIV-infected cells can drive the expression of an anti-SIV pol ribozyme and mobilize the vector to transduce neighbouring cells. The anti-SIV pol ribozyme vector was derived from the SIV backbone and contained the 5'- and 3'LTR including transactivation-response, Psi and Rev-responsive elements, thus requiring Tat and Rev and therefore limiting expression to SIV-infected cells. The data presented here show an early reduction in SIV p27 levels in the presence of the anti-SIV pol ribozyme, as well as successful mobilization (vector RNA constituted approximately 17 % of the total virus pool) and spread of the vector containing this ribozyme. These findings provide direct evidence that mobilization of an anti-retroviral SIV gene therapy vector is feasible in the SIV/macaque model.

Base Sequence↗

PubMed2003

Lentiviral vectors pseudotyped with minimal filovirus envelopes increased gene transfer in murine lung.

A human immunodeficiency virus (HIV)-based vector pseudotyped with the Ebola Zaire (EboZ) viral envelope glycoprotein (GP) was recently shown to transduce murine airway epithelia cells in vivo. In this study, the vector was further redesigned to improve gene transfer and also to increase safety. We used mutant EboZ envelopes for pseudotyping, which resulted in higher titers and increased transduction of airway cells in vivo compared to vectors pseudotyped with wild-type EboZ GP. As these envelopes lack regions associated with toxicity of the wild-type EboZ GP, they should also be safer to use for pseudotyping of lentiviral vectors. In addition, lentiviral vectors were created based on feline immunodeficiency virus and shown to have similar efficiency of transduction compared to HIV-based vectors. The creation of lentiviral vectors with highly engineered EboZ envelopes improved the performance of the system and should also increase its safety since only minimal regions of the EboZ envelope, which lack the toxic domain, are used.

Amino Acid Sequence↗

PubMedDate not supplied

Efficient gene transfer to retinal pigment epithelium cells with long-term expression.

PURPOSE: To evaluate the safety and efficiency of feline immunodeficiency virus (FIV) vectors for gene delivery into the mammalian retina. METHODS: A first-generation FIV vector was constructed and administered into rabbit eyes at two different concentrations by intravitreal or subretinal routes. A second-generation FIV vector was also constructed and administered subretinally into both rabbit and rat eyes at the same concentration. After vector administration, eyes were monitored using slit-lamp biomicroscopy, indirect ophthalmoscopy, fundus photography, and electroretinogram. After the rabbits were killed, eye tissues were processed for light microscopy and immunohistochemical analysis. RESULTS: Administration of both first- and second-generation FIV vectors produced transient vitritis and/or papillitis in rabbits, without other pathologic abnormalities. Retinal pigment epithelium (RPE) cells were the predominant cell type transduced in rabbit eyes, but ganglion cells and Muller cells were also transduced. Transduction was confined to the retinal bleb area. The second-generation FIV vector transduced RPE cells much more efficiently than the first-generation vector (95% vs. 4.5%, respectively; P = 0.0015) in rabbit eyes. In contrast, no toxicity was evident over a 24- to 25-month follow-up period after injection of the second-generation FIV vector into rat eyes. Tropism in the rat eye was similar, including RPE and ganglion cells, and the RPE transduction rate was also high (50%). Transgene expression was persistent in both species over the duration of the experiment. CONCLUSION: Second-generation FIV vectors can efficiently transfer genes into RPE cells with resulting long-term expression, properties potentially valuable to gene therapy approaches to some retinal diseases.

Animals↗