Phase 2 gene therapy trial of an anti-HIV ribozyme in autologous CD34+ cells

Ronald T Mitsuyasu, Thomas C Merigan, Andrew Carr, Jerome A Zack, Mark A Winters, Cassy Workman, Mark Bloch, Jacob Lalezari, Stephen Becker, Lorna Thornton, Bisher Akil, Homayoon Khanlou, Robert Finlayson, Robert McFarlane, Don E Smith, Roger Garsia, David Ma, Matthew Law, John M Murray, Christof von Kalle, Julie A Ely, Sharon M Patino, Alison E Knop, Philip Wong, Alison V Todd, Margaret Haughton, Caroline Fuery, Janet L Macpherson, Geoff P Symonds, Louise A Evans, Susan M Pond, David A Cooper, Ronald T Mitsuyasu, Thomas C Merigan, Andrew Carr, Jerome A Zack, Mark A Winters, Cassy Workman, Mark Bloch, Jacob Lalezari, Stephen Becker, Lorna Thornton, Bisher Akil, Homayoon Khanlou, Robert Finlayson, Robert McFarlane, Don E Smith, Roger Garsia, David Ma, Matthew Law, John M Murray, Christof von Kalle, Julie A Ely, Sharon M Patino, Alison E Knop, Philip Wong, Alison V Todd, Margaret Haughton, Caroline Fuery, Janet L Macpherson, Geoff P Symonds, Louise A Evans, Susan M Pond, David A Cooper

Abstract

Gene transfer has potential as a once-only treatment that reduces viral load, preserves the immune system and avoids lifetime highly active antiretroviral therapy. This study, which is to our knowledge the first randomized, double-blind, placebo-controlled, phase 2 cell-delivered gene transfer clinical trial, was conducted in 74 HIV-1-infected adults who received a tat-vpr-specific anti-HIV ribozyme (OZ1) or placebo delivered in autologous CD34+ hematopoietic progenitor cells. There were no OZ1-related adverse events. There was no statistically significant difference in viral load between the OZ1 and placebo group at the primary end point (average at weeks 47 and 48), but time-weighted areas under the curve from weeks 40-48 and 40-100 were significantly lower in the OZ1 group. Throughout the 100 weeks, CD4+ lymphocyte counts were higher in the OZ1 group. This study indicates that cell-delivered gene transfer is safe and biologically active in individuals with HIV and can be developed as a conventional therapeutic product.

Trial registration: ClinicalTrials.gov NCT00074997.

Figures

Figure 1. Ribozyme target site within HIV-1…
Figure 1. Ribozyme target site within HIV-1 genome, schema of gene modified cell manufacture and protocol design
a) The HIV-1 genome is shown together with the target nucleotide sequence. The cleavage site is indicated by an arrow. b) HIV-1 positive individuals received 30μg/kg/day G-CSF over 5 days. Peripheral blood stem cells were collected by large volume apheresis, using either manual or automatic settings, on days 4 and 5. CD34+ cells were selected and cultured in the presence of Stem Cell Factor and Megakaryocyte Growth & Differentiation Factor (SCF/MGDF) for 30-36 hours to recruit cells into cell cycle in preparation for retroviral transduction with OZ1 or placebo in the presence of RetroNectin, and SCF/MGDF. On Day 8, the gene modified cell product was washed, prepared for infusion and tested for purity, potency and sterility prior to infusion. All participants received their autologous cell product as per the randomization. c) Participants continued HAART for 24 weeks post infusion before entering a four-week treatment interruption (week 24-28) which was intended to apply selective pressure on any OZ1 containing cells. The analytical treatment interruption commenced after week 40 post infusion. Assessments were conducted weekly during the analytical treatment interruption until week 48 and then monthly until either the resumption of HAART or week 100. Participants were advised to recommence HAART if the protocol defined viral load limits were reached: 500,000 copies/ml (up to week 48) and 100,000 copies/ml (week 48-100). HAART was also recommenced if protocol defined limits for CD4+ lymphocyte counts (

Figure 2. Patient Disposition

At screening, a…

Figure 2. Patient Disposition

At screening, a total of 28 patients were excluded; 21 did…

Figure 2. Patient Disposition
At screening, a total of 28 patients were excluded; 21 did not meet eligibility criteria and 7 were excluded for other reasons including intervening adverse events. Of the 78 participants enrolled in the study, 2 were withdrawn prior to randomization and 2 after randomization due to cell processing failures. 3 participants were lost to follow up prior to the primary efficacy endpoint. A total of 9 participants did not enter the analytic treatment interruption.

Figure 3. HIV-1 Viral Load: Intention-to-Treat Population

Figure 3. HIV-1 Viral Load: Intention-to-Treat Population

The mean log 10 viral load shown here…

Figure 3. HIV-1 Viral Load: Intention-to-Treat Population
The mean log10 viral load shown here was determined using real time assay results. As participants recommenced HAART at different times, the n values at the different time points have been included. PCR sequence data of the HIV-1 protease and reverse transcriptase genes were used to generate a virtual phenotype. Similar data was seen for the per-protocol population Only one participant with a mutation associated with resistance to protease inhibitors failed to suppress HIV-1 replication after the recommencement of HAART. screening visit is indicated as scr, ●---●---● OZ1 group, ○- -○- -○ Control group

Figure 4. T lymphocyte counts over time

Figure 4. T lymphocyte counts over time

T lymphocyte counts are presented as a mean…

Figure 4. T lymphocyte counts over time
T lymphocyte counts are presented as a mean percentage of CD3+ T-lymphocytes for each treatment group. For panels A & B, only patients who did not resume HAART from week 40 are included in the analysis. An additional Supplementary Figure 4 showing CD4+ T lymphocytes as percentage of CD3+ T-lymphocytes: Intention-To-Treat Population is in the Supplementary Material. A) Change in CD4 percentage from baseline at screening: Intention-To-Treat Population, B) Change in CD8 percentage from baseline at screening: Intention-To-Treat Population, and C) CD4+ T-lymphocytes as percentage of CD3+ T-lymphocytes only for participants who resumed HAART prior to or at week 52. (At week 100, n=10 in OZ1 treatment group, n=9 in the Control group). The dotted line in the figure represents the baseline percentage. ●---●---● OZ1 group, ○- -○- -○ Control group

Figure 4. T lymphocyte counts over time

Figure 4. T lymphocyte counts over time

T lymphocyte counts are presented as a mean…

Figure 4. T lymphocyte counts over time
T lymphocyte counts are presented as a mean percentage of CD3+ T-lymphocytes for each treatment group. For panels A & B, only patients who did not resume HAART from week 40 are included in the analysis. An additional Supplementary Figure 4 showing CD4+ T lymphocytes as percentage of CD3+ T-lymphocytes: Intention-To-Treat Population is in the Supplementary Material. A) Change in CD4 percentage from baseline at screening: Intention-To-Treat Population, B) Change in CD8 percentage from baseline at screening: Intention-To-Treat Population, and C) CD4+ T-lymphocytes as percentage of CD3+ T-lymphocytes only for participants who resumed HAART prior to or at week 52. (At week 100, n=10 in OZ1 treatment group, n=9 in the Control group). The dotted line in the figure represents the baseline percentage. ●---●---● OZ1 group, ○- -○- -○ Control group

Figure 4. T lymphocyte counts over time

Figure 4. T lymphocyte counts over time

T lymphocyte counts are presented as a mean…

Figure 4. T lymphocyte counts over time
T lymphocyte counts are presented as a mean percentage of CD3+ T-lymphocytes for each treatment group. For panels A & B, only patients who did not resume HAART from week 40 are included in the analysis. An additional Supplementary Figure 4 showing CD4+ T lymphocytes as percentage of CD3+ T-lymphocytes: Intention-To-Treat Population is in the Supplementary Material. A) Change in CD4 percentage from baseline at screening: Intention-To-Treat Population, B) Change in CD8 percentage from baseline at screening: Intention-To-Treat Population, and C) CD4+ T-lymphocytes as percentage of CD3+ T-lymphocytes only for participants who resumed HAART prior to or at week 52. (At week 100, n=10 in OZ1 treatment group, n=9 in the Control group). The dotted line in the figure represents the baseline percentage. ●---●---● OZ1 group, ○- -○- -○ Control group
Figure 2. Patient Disposition
Figure 2. Patient Disposition
At screening, a total of 28 patients were excluded; 21 did not meet eligibility criteria and 7 were excluded for other reasons including intervening adverse events. Of the 78 participants enrolled in the study, 2 were withdrawn prior to randomization and 2 after randomization due to cell processing failures. 3 participants were lost to follow up prior to the primary efficacy endpoint. A total of 9 participants did not enter the analytic treatment interruption.
Figure 3. HIV-1 Viral Load: Intention-to-Treat Population
Figure 3. HIV-1 Viral Load: Intention-to-Treat Population
The mean log10 viral load shown here was determined using real time assay results. As participants recommenced HAART at different times, the n values at the different time points have been included. PCR sequence data of the HIV-1 protease and reverse transcriptase genes were used to generate a virtual phenotype. Similar data was seen for the per-protocol population Only one participant with a mutation associated with resistance to protease inhibitors failed to suppress HIV-1 replication after the recommencement of HAART. screening visit is indicated as scr, ●---●---● OZ1 group, ○- -○- -○ Control group
Figure 4. T lymphocyte counts over time
Figure 4. T lymphocyte counts over time
T lymphocyte counts are presented as a mean percentage of CD3+ T-lymphocytes for each treatment group. For panels A & B, only patients who did not resume HAART from week 40 are included in the analysis. An additional Supplementary Figure 4 showing CD4+ T lymphocytes as percentage of CD3+ T-lymphocytes: Intention-To-Treat Population is in the Supplementary Material. A) Change in CD4 percentage from baseline at screening: Intention-To-Treat Population, B) Change in CD8 percentage from baseline at screening: Intention-To-Treat Population, and C) CD4+ T-lymphocytes as percentage of CD3+ T-lymphocytes only for participants who resumed HAART prior to or at week 52. (At week 100, n=10 in OZ1 treatment group, n=9 in the Control group). The dotted line in the figure represents the baseline percentage. ●---●---● OZ1 group, ○- -○- -○ Control group
Figure 4. T lymphocyte counts over time
Figure 4. T lymphocyte counts over time
T lymphocyte counts are presented as a mean percentage of CD3+ T-lymphocytes for each treatment group. For panels A & B, only patients who did not resume HAART from week 40 are included in the analysis. An additional Supplementary Figure 4 showing CD4+ T lymphocytes as percentage of CD3+ T-lymphocytes: Intention-To-Treat Population is in the Supplementary Material. A) Change in CD4 percentage from baseline at screening: Intention-To-Treat Population, B) Change in CD8 percentage from baseline at screening: Intention-To-Treat Population, and C) CD4+ T-lymphocytes as percentage of CD3+ T-lymphocytes only for participants who resumed HAART prior to or at week 52. (At week 100, n=10 in OZ1 treatment group, n=9 in the Control group). The dotted line in the figure represents the baseline percentage. ●---●---● OZ1 group, ○- -○- -○ Control group
Figure 4. T lymphocyte counts over time
Figure 4. T lymphocyte counts over time
T lymphocyte counts are presented as a mean percentage of CD3+ T-lymphocytes for each treatment group. For panels A & B, only patients who did not resume HAART from week 40 are included in the analysis. An additional Supplementary Figure 4 showing CD4+ T lymphocytes as percentage of CD3+ T-lymphocytes: Intention-To-Treat Population is in the Supplementary Material. A) Change in CD4 percentage from baseline at screening: Intention-To-Treat Population, B) Change in CD8 percentage from baseline at screening: Intention-To-Treat Population, and C) CD4+ T-lymphocytes as percentage of CD3+ T-lymphocytes only for participants who resumed HAART prior to or at week 52. (At week 100, n=10 in OZ1 treatment group, n=9 in the Control group). The dotted line in the figure represents the baseline percentage. ●---●---● OZ1 group, ○- -○- -○ Control group

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