Construction of a Minimal HIV-1 Variant that Selectively Replicates in Leukemic Derived T-Cell Lines: Towards a New Virotherapy Approach

Jeeninga, Rienk E.; Jan, Barbara; Linden, Birgit van der; Berg, Henk van den; Berkhout, Ben

doi:10.1158/0008-5472.can-04-4280

Cited by 19 publications

(22 citation statements)

References 65 publications

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“…We used less supernatant in subsequent passages, from 100 l in the second passage to a minimum of 10 l. Cells and supernatant samples were taken at regular time points and stored at Ϫ70°C. Cell culturing, transfections, and CA-p24 determination were performed as previously reported (5,24). Proviral DNA isolation, PCR amplification, and sequencing.…”

Section: Methodsmentioning

confidence: 99%

Selection of T1249-Resistant Human Immunodeficiency Virus Type 1 Variants

Eggink

Baldwin

Deng

et al. 2008

J Virol

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Human immunodeficiency virus type 1 (HIV-1) entry is an attractive target for therapeutic intervention. Two drugs that inhibit this process have been approved: the fusion inhibitor T20 (enfuvirtide [Fuzeon]) and, more recently, the CCR5 blocker maraviroc (Selzentry). T1249 is a second-generation fusion inhibitor with improved antiviral potency compared to the first-generation peptide T20. We selected T1249-resistant HIV-1 variants in vitro by serial virus passage in the presence of increasing T1249 doses after passage with wild-type and T20-resistant variants. Sequence analysis revealed the acquisition of substitutions within the HR1 region of the gp41 ectodomain. The virus acquired mutations of residue V38 to either E or R in 10 of 19 cultures. Both E and R at position 38 were confirmed to cause resistance to T1249, as well as cross-resistance to T20 and C34, but not to the third-generation fusion inhibitor T2635. We also observed substitutions at residues 79 and 90 (Q79E and K90E), which provide modest resistance to T1249 and, interestingly, T2635. Thus, the gp41 amino acid position implicated in T20 resistance (V38 replaced by A, G, or W) is also responsible for T1249 resistance (V38 replaced by E, R, or K). These results indicate that T20 and T1249 exhibit very similar inhibition modes that call for similar but not identical resistance mutations. All T1249-resistant viruses with changes at position 38 are cross resistant to T20, but not vice versa. Furthermore, substitutions at position 38 do not provide resistance to the third-generation inhibitor T2635, while substitution at positions 79 and 90 do, suggesting different resistance mechanisms.

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Section: Methodsmentioning

confidence: 99%

Selection of T1249-Resistant Human Immunodeficiency Virus Type 1 Variants

Eggink

Baldwin

Deng

et al. 2008

J Virol

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“…Cellular debris was removed by centrifugation for 5 min at 1200 rpm. Production of lentiviral vector particles was determined by CA-p24 ELISA as previously described (Jeeninga et al 2005). Capsid values were corrected for between-session variation (Ruijter et al 2006).…”

Section: Lentiviral Vector Production and Transductionmentioning

confidence: 99%

Titers of lentiviral vectors encoding shRNAs and miRNAs are reduced by different mechanisms that require distinct repair strategies

Liu¹,

Vink²,

Westerink³

et al. 2010

RNA

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RNAi-based gene therapy is a powerful approach to treat viral infections because of its high efficiency and sequence specificity. The HIV-1-based lentiviral vector system is suitable for the delivery of RNAi inducers to HIV-1 susceptible cells due to its ability to transduce nondividing cells, including hematopoietic stem cells, and its ability for stable transgene delivery into the host cell genome. However, the presence of anti-HIV short hairpin RNA (shRNA) and microRNA (miRNA) cassettes can negatively affect the lentiviral vector titers. We show that shRNAs, which target the vector genomic RNA, strongly reduced lentiviral vector titers but inhibition of the RNAi pathway via saturation could rescue vector production. The presence of miRNAs in the vector RNA genome (sense orientation) results in a minor titer reduction due to Drosha processing. A major cause for titer reduction of miRNA vectors is due to incompatibility of the cytomegalovirus promoter with the lentiviral vector system. Replacement of this promoter with an inducible promoter resulted in an almost complete restoration of the vector titer. We also showed that antisense poly(A) signal sequences can have a dramatic effect on the vector titer. These results show that not all sequences are compatible with the lentiviral vector system and that care should be taken in the design of lentiviral vectors encoding RNAi inducers.

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“…This shows that lentiviral genomes bearing alternative Env glycoproteins are capable of replicating in vitro. It remains to be determined, though, how such replication-competent vectors will perform in other cells types, and how the lack of accessory proteins may affect the replication of such artificial RCLs in other cell types, including primary cells ( Jeeninga et al, 2005( Jeeninga et al, , 2006.…”

Section: Discussionmentioning

confidence: 99%

Analysis of Partial Recombinants in Lentiviral Vector Preparations

Kuate

Marino²,

Reiser³

2014

Human Gene Therapy Methods

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The presence of replication-competent lentivirus (RCL) in lentiviral vector preparations is a major safety concern for clinical applications of such vectors. RCL are believed to emerge from rare recombinant vector genomes that are referred to as partial recombinants or Psi-Gag recombinants. To quantitatively determine the fraction of partial recombinants in lentiviral vector preparations and to analyze them at the DNA sequence level, we established a drug selection assay involving a lentiviral packaging construct containing a drug-resistance gene encoding blasticidin (BSD) resistance. Upon transduction of target cells, the BSD resistance gene confers BSD resistance to the transduced cells. The results obtained indicate that there were up to 156 BSD-resistant colonies in a total of 10 6 transducing vector particles. The predicted recombination events were verified by polymerase chain reaction using genomic DNA obtained from BSD-resistant cell clones and by DNA sequence analysis. In an attempt to reduce the emergence of partial recombinants, sequence overlaps between the packaging and the vector constructs were reduced by substituting the Rev response element (RRE) present in the vector construct using a heterologous RRE element derived from simian immunodeficiency virus (SIVmac 239 ). The results obtained showed that a reduction of sequence overlaps resulted in an up to sevenfold reduction of the frequency of BSD-resistant colonies, indicating that the capacity to form partial recombinants was diminished.

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Construction of a Minimal HIV-1 Variant that Selectively Replicates in Leukemic Derived T-Cell Lines: Towards a New Virotherapy Approach

Cited by 19 publications

References 65 publications

Selection of T1249-Resistant Human Immunodeficiency Virus Type 1 Variants

Selection of T1249-Resistant Human Immunodeficiency Virus Type 1 Variants

Titers of lentiviral vectors encoding shRNAs and miRNAs are reduced by different mechanisms that require distinct repair strategies

Analysis of Partial Recombinants in Lentiviral Vector Preparations

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