Inhibition of Moloney murine leukemia virus-induced leukemia in transgenic mice expressing antisense RNA complementary to the retroviral packaging sequences.

Abstract

Recombinant plasmids pLP/as and pCP*as were constructed by positioning the Moloney murine leukemia virus (M-MuLV) proviral packaging (at) sequences in reverse orientation under the transcriptional regulation of lymphotropic promoter/regulatory elements from the M-MuLV long terminal repeat or the cytomegalovirus immediate-early region. Linear fragments containing the antisense 'I' and the appropriate transcriptional regulatory sequences from these plasmids were introduced into the mouse germ line by zygote microinjection. The chromosomal integration, germ-line transmission, and lymphocyte-directed expression of the antisense ## RNA were confirmed. Control (nontransgenic) and transgenic mice containing either the pLPqas or the pCPaas sequences were infected with M-MuLV on the day of birth and assayed for signs ofleukemia between 12 and 14 weeks of age with standard assay procedures. While 31% (11 of 36) of the control, nontransgenic, mice developed leukemia, none of the antisense 4i transgenic mice developed any symptoms of leukemia. The pCPabas sequences were also introduced into mouse NIH 3T3 cells and stably transformed cell lines were isolated. When infected with M-MuLV these cells were shown to produce virus devoid of packaged viral RNA.Retroviruses require only a very limited number of genes and genetic regulatory sequences to complete their cycle of infection in host cells or organisms (1-4). This remarkable molecular efficiency partially explains why retroviruses such as human immunodeficiency virus (HIV) act so effectively as pathogens, but may also suggest targets for molecular attenuation of retroviral replication in host organisms. Among the most stringent requirements for retroviral replication are cis-acting viral genomic sequences necessary for the specific encapsidation of genomic viral RNA molecules into virus particles (4-10). These packaging (4i) sequences have been identified and used extensively as the basis of retroviral vectors designed for gene transfer (10-13). Results from these combined studies suggest that interference with the specific interactions between these 4i sequences and virion capsid proteins will block the retroviral replication cycle.The use of antisense RNA to inhibit RNA function within cells and whole organism has provided a valuable molecular method (14-16). Antisense RNA can bind in a highly specific manner to complementary sequences, blocking the ability of the bound RNA to be processed or translated or, possibly, even to interact with sequence-specific binding proteins (17)(18)(19)(20). To test the efficacy of antisense RNA complementary to the sequences of a murine retrovirus in blocking retroviral replication, transgenic mice expressing RNA sequences complementary to the q, sequences of Moloney murine leukemia virus (M-MuLV) were produced and then challenged with this virus. MATERIALS AND METHODSPlasmids. Recombinant plasmids pLPqias and pCP/as were constructed as shown in Fig. 1. Plasmid pLJ (21) was cleaved with Sma I and the 540-bp Sma I fragment containi...

Inhibition of Moloney murine leukaemia virus by a retroviral vector, LNL6, carrying ribozymes targeted to the 5' non-coding sequence.

“…Even though the antisense molecules used are relatively short, the presence of the catalytic core, a structure conserved through evolution (Haseloff & Gerlach, 1988), may result in a more stable and effective antisense molecule. This result is also consistent with the published observation of an antisense effect for a longer 540 base antisense sequence to the corresponding MoMLV Ψ region in transgenic mice (Han et al, 1991).…”

“…Interruption of this interaction has been shown to block the retroviral replication cycle and deletion mutants in this region are not packaged into virions (Sullenger et al, 1990). In previous studies (Han et al, 1991), antisense RNA complementary to the packaging region has been shown to inhibit virus replication in cell lines, and transgenic mice expressing the same antisense RNA to this region showed a reduction in both viraemia and the induction of leukaemia (Han et al, 1991).…”

Therapeutic Effect of a Gag-Nuclease Fusion Protein against Retroviral Infection In Vivo

“…The hCMV-E/P (8) appeared most useful for our experiments, because this control element facilitated transgene expression in 28 tissues analyzed (13). Moreover, hCMV-driven transgenes were reproducibly shown to be highly expressed in spleen, thymus, and other lymphatic tissues (13,15,45).…”

“…This is the first transgenic mouse model for a proteinbased antiviral strategy. (A murine in vivo nucleic-acid-based antiviral strategy was described previously [15]. )…”

“…This is the first transgenic mouse model for a proteinbased antiviral strategy. (A murine in vivo nucleic-acid-based antiviral strategy was described previously [15].) gene cassette from pGN1595, flanked by XbaI and SalI restriction sites, was inserted into the multiple cloning site of pGN1717, which separates the hCMV-P/E element from the SV40 poly(A) signal, leading to pGN1743.…”