Co-packaging of non-vector RNAs generates replication-defective retroviral vector particles: a novel approach for blocking retrovirus replication

Abstract

A Moloney murine leukemia virus (MoMuLV)-derived packaging retroviral vector, pUCMoTN-PR3, was previously developed in which the packaging (psi) signal was cloned within the 5'-long terminal repeat (LTR) U3-r and U5 sequences. The MoTN-PR3 vector particles released from a transfected packaging cell line contain RNAs with r-psi-U5 sequences at the 5'-end and U3-r sequences at the 3'-end. Upon infection, these vector particles can efficiently transduce the neomycin phosphotransferase (neo) gene to the target cells. The structure of the proviral DNA synthesized in these cells was shown to contain modified 5'- and 3'-LTRs with U3-r-psi-U5 sequences, indicating that this vector can undergo reverse transcription and integration. Analysis of psi signal-containing RNAs revealed that in addition to vector RNA transcribed from the MoMuLV 5'-LTR promoter, readthrough neo RNA transcribed from the internal herpes simplex virus (HSV) thymidine kinase (tk) promoter and cellular RNAs transcribed from the MoMuLV 3'-LTR promoter are produced. Of these, the downstream cellular RNAs are also packaged within the vector particles. These vector particles containing the vector and non-vector RNAs carrying the MoMuLV psi signal are non-infectious. It is proposed that intracellular expression of packageable non-viral RNAs may represent an effective strategy for inhibiting animal and plant virus replication.

A combination anti-HIV-1 gene therapy approach using a single transcription unit that expresses antisense decoy and sense RNAS and trans-dominant negative mutant gag and env proteins

“…HIV-1 TAR and RRE, which are present on both GE and GmEm mRNAs, should confer partial (but similar) inhibition of HIV-1 replication by providing competitive protein binding sites and/or by sequestering key cellular factors (1). The HIV-1 Ψ e signal present in GE mRNA should inhibit HIV-1 replication by competing with HIV-1 RNA for packaging within the progeny virus (8,33). However, the HIV-1 packaging signal sequences present within the GmEm mRNA (due to a 181-bp deletion within the TDM gag coding region) may not allow this RNA to efficiently compete with HIV-1 RNA for packaging within the virions.…”

Genetic-Based Therapies to Select Nonpathogenic Variants of HIV-1

“…However, mobilization, while initially viewed as less than favorable, has been suggested to be a suitable trait that can suppress viral pathogenesis [54]. Moreover, lentiviral-based vector mobilization allows for competition with the wild-type virus for packaging as well as trans elements and can, in fact, spread the therapeutic vector to the same target cells infected by the virion (Figure 3) as well as reduce the infectivity of the wild-type virus [55]. Nonetheless, the risk of lentiviral vector recombination persists [56,57].…”

Current develoments and future prospects for HIV gene therapy using interfering RNA-based strategies

“…Lack of inhibition was likely due to the inability of this RNA to efficiently compete with HIV-1 RNA for packaging within the progeny virus; inclusion of a 1 kb region within the env coding region (encompassing RRE) has been shown to be required for efficient viral RNA packaging (181). A single copy retroviral vector was then designed to express a 1.8 kb sense RNA containing the 5' untranslated region (which includes TAR) and the Psi-e signal (which includes RRE) (108).…”