The acquired immune deficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV) is one of the acute problems of today's world that should be considered. Given the promising results of the drugs used to treat this disease, they are not without problems due to side effects such as drug-related toxicity. In addition, drug failure due to virus resistance is common in long-term antiviral chemotherapy [1–3]. Thus, there is a need to develop new alternative therapies to cure patients with chronic HIV infection. One of these advanced therapies is genetic-based approaches such as the use of suicide genes [4, 5], dominant-negative proteins [6, 7], and oligonucleotide-based materials [8, 9]. Some of these therapies have shown promising results in inhibiting HIV replication and some of those have continued to a clinical trial. Given the main problem in controlling HIV infection is the ability of the virus to resist drugs and avoid treatment, so the need to design strategies to control virus replication seems essential. These strategies can include targeting the different viral products in early and late steps of the virus life cycle, designing RNAs interference (RNAi) against the conserved regions on virus transcripts, or a combination of the two strategies. These strategies have shown that they can remarkably reduce the escape of the mutated virus [10, 11].
Posttranscriptional silencing by RNAi has been used in many studies to develop new approaches in antiviral therapy [12, 13]. One of the major RNAis is small interfering RNA (siRNA), which is a double-stranded RNA molecule comprising 22-23 nucleotides and two nucleotide protrusions at both ends of 3´ and is modified by the Dicer complex. Dicer is an endoribonuclease that actives the RNA-induced silencing complex [14, 15]. The siRNA can be transfected as synthetic or as an expression plasmid with sense and antisense strands separately. Another strategy to construct siRNA is short-stranded RNA (shRNA), a 50-60 nucleotide single-stranded RNA that is modified to a siRNA molecule [16–18].
One of the best approaches in gene therapy for HIV-infected cells is to use a selective inducible promoter for the expression of inhibitory genes. In some previous studies, some of these promoters have been developed, including the native HIV long terminal repeat (LTR) [19–23], chimeric LTR-hsp [22, 23], and chimeric CK-TAR [24, 25]. All these promoters are induced by trans-activating regulatory protein (Tat) produced by HIV , thus they express their controlled genes specifically in HIV-infected cells. The CK-TAR promoter can be safer compared to native LTR and LTR-hsp because it lacks the risk of LTR remodeling. However, in an shRNA expression cassette, the CK-TAR promoter cannot generate an shRNA site with the desired transcription start site. Also, It had a relative amount of basal expression in the absence of Tat protein that reduces its eligibility in selective expression . To overcome these risks, we recently developed a Tat-inducible promoter named the CkRhsp, with neutralized LTR mobilization and a favorable transcriptional start site to express an anti-HIV shRNA .
In the last decade, Adenoviral (Ad) vectors have been frequently used for gene delivery. They have been well studied and can be produced to high titer. The Ad vectors also efficiently express a transgene in both dividing and non-dividing cells and can be conserved in cells as episomal form [28–30]. The results of almost all clinical trials with this vector indicated that Ad vectors are safe and well-tolerated . Despite the versatility of recombinant Ad vectors in gene therapy approaches, the diversity in the use of these vectors in gene transfer applications is limited due to their semi-specific tropism and is best suited for a specific organ rather than a specific cell type . One strategy to overcome this tropism restriction could be to modify the domains of the proteins involved in binding the virus to the cell through which the vector is allowed to interact with the receptors of a particular cell. Ads bind to a specific receptor in eukaryotic cells via the Knob domain at the end of their fiber protein. Thus, maybe protein engineering of fiber protein will alter the natural tropism of Ads [33, 34].
In this study, we decided to design an Ad5 vector with a specific tropism to CD4-positive cells. Thus, we replaced the Knob region of the Ad5 fiber protein with the extracellular domain of the HIV-1 envelope protein. To obtain a restricted expression system for HIV-infected cells, we used a TAT-inducible promoter called CkRhsp to express two anti-HIV-1 shRNAs, as we have reported previously .