Gene Therapy in HIV‐Infected Cells to Decrease Viral Impact by Using an Alternative Delivery Method

Abstract

The ability of dendrimer 2G-[Si{O(CH(2))(2)N(Me)(2) (+)(CH(2))(2)NMe(3) (+)(I(-))(2)}](8) (NN16) to transfect a wide range of cell types, as well as the possible biomedical application in direct or indirect inhibition of HIV replication, was investigated. Cells implicated in HIV infection such as primary peripheral blood mononuclear cells (PBMC) and immortalized suspension cells (lymphocytes), primary macrophages and dendritic cells, and immortalized adherent cells (astrocytes and trophoblasts) were analyzed. Dendrimer toxicity was evaluated by mitochondrial activity, cell membrane rupture, release of lactate dehydrogenase, erythrocyte hemolysis, and the effect on global gene expression profiles using whole-genome human microarrays. Cellular uptake of genetic material was determined using flow cytometry and confocal microscopy. Transfection efficiency and gene knockdown was investigated using dendrimer-delivered antisense oligonucleotides and small interfering RNA (siRNA). Very little cytotoxicity was detected in a variety of cells relevant to HIV infection and erythrocytes after NN16 dendrimer treatment. Imaging of cellular uptake showed high transfection efficiency of genetic material in all cells tested. Interestingly, NN16 further enhanced the reduction of HIV protein 24 antigen release by antisense oligonucleotides due to improved transfection efficiency. Finally, the dendrimer complexed with siRNA exhibited therapeutic potential by specifically inhibiting cyclooxygenase-2 gene expression in HIV-infected nervous system cells. NN16 dendrimers demonstrated the ability to transfect genetic material into a vast array of cells relevant to HIV pathology, combining high efficacy with low toxicity. These results suggest that NN16 dendrimers have the potential to be used as a versatile non-viral vector for gene therapy against HIV infection.

Potential inhibition of HIV-1 encapsidation by oligoribonucleotide–dendrimer nanoparticle complexes

“…7% and 1.9%, respectively) (Figure 4). By comparison, in other studies, transfection efficiencies of 36% and 90% were reported with the use of a second-generation carbosilane dendrimer in primary T lymphocytes 33 and SupT1 lymphocytes, 34 respectively. The RNA alone was taken up by 9.3% of cells.…”

“…The slides were fixed with paraformaldehyde and incubated for 10 minutes with Fluoroshield™ with 4′,6-diamidino-2-phenylindole (DAPI) stain and then viewed and photographed under confocal microscope, ZEISS Confocal LSM 710 (Zeiss, Oberkochen, Germany). 34 …”

“…31,32 Nonetheless, there have been reports of successful transfection of lymphocytes using carbosilane dendrimers. 33,34 This prompted us to investigate the potential of a carbosilane dendrimer to deliver the SL3 oligoribonucleotide decoy into CD4 + lymphocytes, thereby inhibiting the replication of HIV.…”

Anti-HIV-1 nanotherapeutics: promises and challenges for the future

“…60 Nanoparticles can improve delivery of antigens to enhance the immune response and can encapsulate antigens in their core from which antigen-presenting cells (APCs) can process and present antigens to CD4 + and CD8 + T cells or facilitate B cells to generate humoral responses. [92][93][94][95] Various polymeric and lipid-based nanoparticles have been used to deliver DNA-, protein-, or peptide-based antigens in vivo, which elicit strong cellular, humoral, and mucosal immune responses. However, no effective vaccine strategy has emerged in spite of promising preliminary results.…”

“…Thus, γ-PGA nanoparticles encapsulating various antigens may have great potential as novel and efficient protein-based vaccines against HIV-1 infection. 11,27,95 Several liposome-based systems using a variety of HIV-1 antigenic peptides such as gp160, gp41, gp120, p24, tat, gag, env, and rev have been evaluated for HIV vaccine delivery, however none of them have been able to elicit a broad neutralizing antibody response. 32 …”