The development of retroviral vectors that target specific cell types could have important implications for the design of gene therapy strategies. A chimeric protein containing the polypeptide hormone erythropoietin and part of the env protein of ecotropic Moloney murine leukemia virus was engineered into the virus. This murine virus became several times more infectious for murine cells bearing the erythropoietin receptor, and it also became infectious for human cells bearing the erythropoietin receptor. This type of tissue-specific targeting by means of ligand-receptor interactions may have broad applications to a variety of gene delivery systems.

Whether long interspersed element-1 (L1 or LINE-1) retrotransposition can occur in quiescent, nondividing, and͞or terminally differentiated somatic cells has remained an unanswered fundamental question in human genetics. Here, we used a ubiquitously active phosphoglycerate kinase-1 promoter to drive the expression of a highly active human L1 element from an adenovirus-L1 hybrid vector. This vector system achieved retrotransposition in up to 91% of actively growing immortalized cells, and we demonstrated that L1 retrotransposition can be suppressed by the reverse transcriptase inhibitor 3 -azido-3 -deoxythymidine. This adenovirus vector enabled efficient delivery of the L1 element into differentiated primary human somatic cells and G 1͞S-arrested cells, resulting in retrotransposition in both cases; however, it was not detected in G 0-arrested cells. Thus, these data indicate that L1 retrotransposition can occur in nondividing somatic cells.adenovirus ͉ LINE-1 ͉ quiescent cell ͉ hybrid vector R etrotransposons are mobile elements that insert into new genomic locations by reverse transcription of an RNA intermediate. Human long interspersed element-1 (L1) elements (1, 2) are non-LTR retrotransposons that comprise Ͼ17% of the human genome. The vast majority (Ͼ99%) of L1s are inactive because of point mutations, truncations, and other rearrangements; however, it is estimated that the average human diploid genome contains Ϸ80-100 retrotransposition-competent (RC)-L1s (3). RC-L1s have played and continue to play a significant role in shaping the genome through insertional mutagenesis, nonallelic recombination, and by trans mobilization of non-L1 RNAs (2, 4, 5).L1 retrotransposition requires transcription of L1 RNA, its transport to the cytoplasm, and translation of its two ORFs (ORF1 and ORF2). Both L1-encoded proteins (ORF1p and ORF2p) are thought to preferentially associate with their own encoding RNA (''cis preference'') to form a ribonucleoprotein particle (RNP) (6, 7), which is a proposed retrotransposition intermediate (8,9). The L1 RNP must access the nucleus, where the L1 endonuclease cleaves genomic DNA at a degenerate consensus sequence (5Ј-TTTT͞A and variant sequences) to liberate a 3Ј hydroxyl residue that is subsequently used by the L1 reverse transcriptase (RT) as a primer to copy the L1 sequence in situ, a process termed ''targetprimed reverse transcription'' (2, 10). The resultant L1 cDNA then is joined to target DNA, leading to typical L1 structural hallmarks [5Ј truncations and͞or internal inversions, 3Ј poly (A) tail, and target-site duplications (TSDs)]. Although a putative nucleolar localization signal has been identified in ORF2p (11), it still remains unclear whether the L1 RNP crosses an intact nuclear membrane or whether its entry requires mitotic nuclear envelope breakdown.We previously developed a hybrid vector system consisting of a high-capacity, helper-dependent adenovirus vector encoding a human RC-L1 element (L1.3) tagged with a neomycin-resistance (neo R ) retrotransposition indicator ...

Patients with the most common and aggressive form of high-grade glioma, glioblastoma multiforme, have poor prognosis and few treatment options. In 2 immunocompetent mouse brain tumor models (CT26-BALB/c and Tu-2449-B6C3F1), we showed that a nonlytic retroviral replicating vector (Toca 511) stably delivers an optimized cytosine deaminase prodrug activating gene to the tumor lesion and leads to long-term survival after treatment with 5-fluorocytosine (5-FC). Survival benefit is dose dependent for both vector and 5-FC, and as few as 4 cycles of 5-FC dosing after Toca 511 therapy provides significant survival advantage. In the virally permissive CT26-BALB/c model, spread of Toca 511 to other tissues, particularly lymphoid tissues, is detectable by polymerase chain reaction (PCR) over a wide range of levels. In the Tu-2449-B6C3F1 model, Toca 511 PCR signal in nontumor tissues is much lower, spread is not always observed, and when observed, is mainly detected in lymphoid tissues at low levels. The difference in vector genome spread correlates with a more effective antiviral restriction element, APOBEC3, present in the B6C3F1 mice. Despite these differences, neither strain showed signs of treatment-related toxicity. These data support the concept that, in immunocompetent animals, a replicating retroviral vector carrying a prodrug activating gene (Toca 511) can spread through a tumor mass, leading to selective elimination of the tumor after prodrug administration, without local or systemic pathology. This concept is under investigation in an ongoing phase I/II clinical trial of Toca 511 in combination with 5-FC in patients with recurrent high-grade glioma ( NCT01156584).

Achieving therapeutically efficacious levels of gene transfer in tumors has been a major obstacle for cancer gene therapy using replication-defective virus vectors. Recently, replicating viruses have emerged as attractive tools for cancer therapy, but generally achieve only transitory tumor regression. In contrast to other replicating virus systems, transduction by replication-competent retrovirus (RCR) vectors is efficient, tumor-selective, and persistent. Correlating with its efficient replicative spread, RCR vector expressing the yeast cytosine deaminase suicide gene exhibited remarkably enhanced cytotoxicity in vitro after administration of the prodrug 5-fluorocytosine. In vivo, RCR vectors replicated throughout preestablished primary gliomas without spread to adjacent normal brain, resulting in profound tumor inhibition after a single injection of virus and single cycle of prodrug administration. Furthermore, stable integration of the replicating vector resulted in persistent infection that achieved complete transduction of ectopic glioma foci that had migrated away from the primary tumor site. Thus, efficient and stable integration of suicide genes represents a unique property of the RCR vector that achieved multiple cycles of synchronous cell killing upon repeated prodrug administration, resulting in chronic suppression of tumor growth and prolonged survival.