In silico modeling indicates the development of HIV-1 resistance to multiple shRNA gene therapy differs to standard antiretroviral therapy

Abstract

Background: Gene therapy has the potential to counter problems that still hamper standard HIV antiretroviral therapy, such as toxicity, patient adherence and the development of resistance. RNA interference can suppress HIV replication as a gene therapeutic via expressed short hairpin RNAs (shRNAs). It is now clear that multiple shRNAs will likely be required to suppress infection and prevent the emergence of resistant virus.

Results: We have developed the first biologically relevant stochastic model in which multiple shRNAs are introduced into CD34+ hematopoietic stem cells. This model has been used to track the production of gene-containing CD4+ T cells, the degree of HIV infection, and the development of HIV resistance in lymphoid tissue for 13 years. In this model, we found that at least four active shRNAs were required to suppress HIV infection/replication effectively and prevent the development of resistance. The inhibition of incoming virus was shown to be critical for effective treatment. The low potential for resistance development that we found is largely due to a pool of replicating wild-type HIV that is maintained in non-gene containing CD4+ T cells. This wild-type HIV effectively out-competes emerging viral strains, maintaining the viral status quo.

Conclusions: The presence of a group of cells that lack the gene therapeutic and is available for infection by wild-type virus appears to mitigate the development of resistance observed with systemic antiretroviral therapy.

Figure 1

Key steps, decision points and probabilities of the 3 D stochastic model. The following parameters were used to determine cell death and replacement, and infection. Cells that do, or do not, contain the integrated gene are referred to as transduced (Tx) or untransduced (UNTx) respectively. Tx or UNTx cells can either be uninfected or infected. (A) The replacement of an infected cell is determined by (1) finding the infected UNTx or Tx cells, (2) identifying the infected dead cells, and (3) replacing them with cells divided from uninfected neighbours or newly matured from the thymus (B) Infection is established by (4) finding an uninfected cell with at least one infected neighbour and determining the protection of the uninfected cell, i.e. is it UNTx or Tx (and with how many shRNAs)? (5) The status of the infected neighbour is used to determine the likely number of virions produced and their sequence. (6) The virion sequence is mutated and recombined as necessary. (7) Cells are infected depending on the infecting viral sequence, any inhibitory shRNA, and chance. (8) The life span of the newly infected cell is randomly assigned and the viral fitness is adjusted according to its mutations/recombinations. Probabilities: P.tx (set at either 0.2 or 0.01): the percentage of Tx CD34+ hematopoietic stem cells (HSC) resulting in this percentage of cells exported from thymus containing gene product. P.neigh (set at 0.99): the replacement by an uninfected neighbour, compared to a cell from the thymus. P.mut (set at 3.4 × 10^-5): the mutation rate per nucleotide. Viral productivity: determined by viral fitness, the transduction state of the infected cell (Tx or UNTx) and the number of mutated sequences. Life span: Poisson distributed with mean 2 days, measured in 12-hourly intervals. P.long (set at 0.0183): probability that an infected cell is long lived.

Figure 2

Increasing the number of shRNAs. Tx and UNTx, infected and uninfected cells are expressed as a percentage of all cells and monitored over 5000 days. Scenarios include; A) The absence of gene therapy B) 6 shRNAs (S1), C) 4 shRNAs (S2) and D) 2 shRNAs (S3). Assumptions for each scenario include 80ⁿ % efficacy (80e), 20% Tx hematopoietic stem cells containing the gene therapeutic (HSC+), 99% fitness (99f) with Class I and II inhibition.

Figure 3

Effect of the number of shRNA, efficacy, marking and level of inhibition on cellular compartments. Cells within each compartment are expressed as a percentage of all cells and monitored over 5000 days. Scenarios include: A) 6 shRNAs (S1), B) 4 shRNAs (S2), C) 2 shRNAs (S3), D) 60ⁿ % efficacy (S4), E) 1% marking (S5) and F) Class II inhibition only (S11). Assumptions for each scenario are indicated where - x = number of shRNA, - e = efficacy, - HSC+ = hematopoietic stem cells transduced to contain the gene therapeutic and - f = fitness.

Figure 4

Effect on the resistance profile in the Tx and UNTx compartments over 5000 days. The percentage of cells, both Tx and UNTx, infected with virus that is wt, or completely resistant to 1 (m = 1) or 2 (m = 2) shRNA and monitored over 5000 days. Scenarios include A) 6 shRNAs (S1), B) 4 shRNAs (S2) and C) 2 shRNAs (S3), D) 60ⁿ % efficacy (S4), E) 50ⁿ % fitness (S6), F) 1% marking and 50ⁿ % fitness (S7) and G) 90% fitness (S10) and H) Class II inhibition only (S11). Assumptions for each scenario are indicated where - × = number of shRNA, - e = efficacy, - HSC+ = hematopoietic stem cells transduced to contain the gene therapeutic and - f = fitness. Populations that were essentially zero were unable to be plotted on a log scale, and are indicated with an appropriate marker placed at the end of the abscissa.

Figure 5

Selection pressures driving the development of resistance. The selection pressures driving resistance in (A) systemic antiretroviral therapy, compared to (B) the bipartite distribution of gene therapy.