to circumvent apobec3 expression in virus - producing cells, which diminishes virus infectivity, a vector containing two overlapping fragments of a3g - d128k was designed that maintained the gene in an inactive form in the virus - producer cells. however, during transduction of target cells, retroviral recombination between the direct repeats reconstituted an active a3g - d128k in 89 % – 98 % of transduced cells. lentiviral vectors that expressed a3g - d128k transduced cd34 hematopoietic stem and progenitor cells with a high efficiency ( > 30 % ). a3g - d128k expression in t cell lines cem, cemss, and pm1 potently inhibited spreading infection of several hiv - 1 subtypes by c - to - u deamination leading to lethal g - to - a hypermutation and inhibition of reverse transcription. sivmac239 and hiv - 2 were not inhibited, since their vifs degraded a3g - d128k. a3g - d128k expression in cem cells potently suppressed hiv - 1 replication for > 3. 5 months without detectable resistant virus, suggesting a high genetic barrier for the emergence of a3g - d128k resistance. because of this, a3g - d128k expression in hiv - 1 target cells is a potential anti-hiv gene therapy approach that could be combined with other therapies for the treatment and functional cure of hiv - 1 infection. treatment for hiv - 1 infection with potent combination antiretroviral therapy ( cart ) can control viral replication and delay onset of aids. however, long - lived latently infected cells can persist and rekindle viral infection upon therapy interruption, resulting in lifelong cart associated with high costs, toxicities, incomplete adherence, and drug resistance. therefore, strategies that can eradicate most infected cells from patients or control viral replication in the absence of cart to achieve a functional cure are needed. the apparent cure of the “ berlin patient ” and recent long - term hiv - 1 remission of another patient by transplantation of hematopoietic stem cells that did not express coreceptor chemok ine receptor 5, or ccr5, have raised hopes of a cure. unfortunately, other cases of allogeneic stem cell transplantation have led to viral rebound5, 6, 7 and / or the emergence of rare pre - existing c - x - c chemok ine receptor type 4, or cxcr4 variants, suggesting that novel strategies are needed to achieve a functional cure for hiv - 1 infection. one potential approach to controlling hiv - 1 replication without cart is to interfere with hiv - 1 ’ s ability to counteract innate host restriction factors, such as the apobec3 family of cytidine deaminases, the tripartite interaction motif 5 alpha protein ( trim5α ), the sterile alpha motif ( sam ) domain and histidine - aspartate ( hd ) domain - containing protein 1 ( samhd1 ), the bst2 / tetherin protein, and the mxb / mx2 protein. 16, 17, 18 restriction factor apobec3g ( a3g ) is a cellular cytidine deaminase that is packaged into newly formed virions and, upon subsequent infection, induces c - to - u deamination of the minus - strand dna during reverse transcription, resulting in lethal g - to - a hypermutation s. a3g can also restrict hiv - 1 through deaminase - independent mechanisms, leading to the inhibition of reverse transcription21, 22, 23 and integration. hiv - 1 has evolved to combat a3g by expressing vif, which targets a3g for polyubiquitination and proteasomal degradation, thereby preventing its virion incorporation. therapeutic strategies based on expressing a vif - resistant a3g mutant in hiv - 1 target cells is an attractive approach to hiv - 1 gene therapy, but efficient delivery of a functional a3g to target cells is technically challenging. viral vectors will inevitably express a3g in the producer cells, and its incorporation into virions will inactivate the vector and the vector - encoded a3g. previous gene therapy studies have proposed to utilize a3g to block hiv - 1 replication by inhibiting a3g ubiquitination, increasing virion incorporation of vif - resistant a3g mutants, expressing an hiv - 1 vif - resistant african green monkey a3g, and using an inducible promoter to block expression of a3g in the producer cells. each of these studies tried to avert the potent inhibitory effects of a3g in the virus - producing cells but faced difficulties in achieving efficient delivery of a3g to target cells. ao et al. used an adeno - associated viral vector system ( aav2 / 5 ) to introduce vif - resistant mutant a3g - p129a into peripheral blood mononuclear cells ( pbmcs ) and macrophages, but potent inhibition of hiv - 1 replication was not observed in pbmcs, perhaps because aav2 / 5 did not efficiently transduce human cd4 t cells. voit et al. inserted a3g - d128k along with a dominant - negative mutant of hiv - 1 rev ( revm10 ) and human / rhesus trim5α by gene editing into the ccr5 locus. although the efficiency of gene delivery was not addressed, expression of a3g - d128k alone was shown to provide the strongest protection ( 100 - to 200 - fold ) from hiv - 1 replication compared to human / rhesus trim5α and revm10. all these approaches have been hampered by low efficiency of transduction, inability to transduce the natural target cells of hiv - 1 infection, and low efficiency of genome editing. we previously described the development of self - activating retroviral vectors for gene therapy using directly repeated nucleot ide sequences. as outlined by the dynamic copy choice model for retroviral recombination, duplicated gene sequences are precisely and ac curate ly deleted at a high efficiency by homologous recombination during the process of reverse transcription. 38, 39, 40, 41, 42, 43 here, we developed a self - activating lentiviral vector that utilized directly repeated sequences of a vif - resistant mutant of a3g ( a3g - d128k ) to prevent expression of a3g - d128k in the lentivector producer cells but reconstituted a functional a3g - d128k in the target cells during retroviral transduction. the results demonstrate that the vectors can be used to efficiently transduce cd4 t cell lines and hematopoietic stem and progenitor cells ( hspcs ). importantly, the in vitro experiments indicated that selection for a3g - d128k - resistant hiv - 1 variants has a high genetic barrier, adding further support to potential anti-hiv gene therapy by the expression of a3g - d128k to control hiv - 1 replication and spread. human cd4 t cells express a3g, which, in the absence of the virally encoded vif, potently inhibits hiv - 1 replication. a3g mutant d128k ( a3g - d128k ) is resistant to hiv - 1 vif - mediated degradation, and its expression in human cd4 t cells should result in potent inhibition of wild - type hiv - 1 replication and spread. lentiviral vectors are one of the most efficient vehicles for delivery of therapeutic genes to human cd4 t cells. however, delivery of a3g - d128k using a traditional lentiviral vector is inefficient, since expression of a3g - d128k in the virus - producing cells results in its virion incorporation, leading to drastic loss of virion infectivity and lethal hypermutation of the therapeutic gene. to prevent expression of a3g - d128k in the lentivector producer cells, we constructed lentiviral vectors that encoded two overlapping fragments of a3g - d128k ( referred to as a3 and 3g; figure 1a ). one copy of the overlapping ∼900 - bp direct repeat is efficiently deleted during reverse transcription by hiv - 1 reverse transcriptase switching templates in the homologous repeats, resulting in the functional reconstitution of a3g - d128k ( figure s1; reviewed by delviks - frankenberry et al. ). briefly, during rna - dependent dna synthesis of the 3 ′ direct repeat ( the “ 3 ” portion of 3g ), the rnase h activity of reverse transcriptase degrades the template rna, which allows the nascent dna strand to anneal to the crna in the 5 ′ direct repeat ( the “ 3 ” portion of a3 ).