Our Approach

Our gene therapy process works by genetically modifying patients’ own cells to shield and strengthen their immune system.

The promise of this approach is to create enduring and perhaps life-long protection for individuals living with a broad range of debilitating and currently incurable diseases.

We have extensive expertise and more than 120 patents in viral vector and non-vector delivery technology, therapeutic product design and manufacturing, which we have developed into a potent gene therapy platform with the potential of broad applications in a wide variety of indications.

Our treatments seek to shield and strengthen patients’ immune system, rather than trying to modify the virus itself.

Outpatient Process:
Using an Individual’s Own Cells with the Objective of Engineering Immunity

Harvest

Separate

Modify

Treat

 
 

Mobilizing Agent

harvest
 
 

(CD4+) T-Cells

 
 

(CD34+) Stem Cells

separate
modify
 
 

Conditioning

treat

Harvest

Separate

Modify

Treat

A mobilizing agent sends cells from the bone marrow into the circulating blood. We then collect components of the blood through a standard machine.

We isolate and purify the target cells collected from the blood: hematopoietic CD34+ stem cells and CD4+ T cells (key elements of the immune system).

We treat the purified cells with the therapeutic vector outside of the body, expand some of them, freeze them and conduct tests prior to release.

We thaw and reinfuse the modified cells after preparing the patient with a conditioning agent intended to make room for the new cell army.

Changing the Paradigm of Stem Cell Gene Therapy

Today

hospital

Calimmune Approach

house
  • In-patient
  • High-dose Conditioning
  • Complicated AE Profile (including sterility/infertility)
  • Patient engraftment variation with risk of missing therapeutic window
  • May require lifetime guarantee
  • Out-patient
  • Low-dose Conditioning
  • Limited AE profile (no sterility or infertility issues)
  • Use of safe registered oral drug to hit therapeutic window
  • Ability to amplify therapy post initial treatement

Select+™

Increases engraftment and enables out-patient treatment

chemoselection

Genetically modify cells to confer 6TG resistance

Stem Cell Gene Therapy Select+
Treatment Setting 14-21 Day Hospitalization Outpatient
Scalability Limited Improves options
ChemoTx Dosing Higher toxic dose Low dose
Engraftment +/- Improves efficiency
Reproductive Impact Causes sterility and infertility None at anticipated dose

Cytegrity™ Efficient, Scalable Proprietary GMP Production System

Establishes a gold standard for lentiviral vector production

Production Transient Transfection Cytegrity
Yield Inconsistent Standardized, large-volume GMP vector (250L+)
RCL risk Significant in large quantities Minimal
Scalability Difficult and costly Simple and cost-efficient
Batch-to-batch consistency Variable High

Vector Technology

Cytegrity™: A Scalable Solution for Gene Therapy

Viral vectors play a fundamental role in gene therapy. They’re the vehicles for delivering new, beneficial genetic material into patients’ cells, and millions of them are used in a single procedure. They are most commonly based on naturally-occurring viruses that have been modified (stripped) of all of the components required to self-replicate and infect additional cells. These viral vectors are ideal for introducing gene therapies.

The traditional process for manufacturing these therapeutic vectors is expensive, labor-intensive and yields small batches. We have exclusively licensed and developed a proprietary cell-based vector manufacturing process that is both reproducible and scalable.

Calimmune’s Cytegrity™ platform is an important step toward advancing the field of gene therapy by realizing its full potential on a commercial scale. Not only does this technology enable gene therapy manufacturing for large patient populations, but also due to its streamlined automation capacity, will significantly lower the cost of production—and ultimately, the cost of treatment.

We are committed to making this technology broadly available to academic, government and corporate partners, and we believe that effective gene therapies should be accessible to all patients who can benefit.

Abstracts

Publications

  • Cell-Delivered Gene Therapy: This Viral Vector Manufacturing Method Could Widen Its Applicability

    Symonds, G. Cell-Delivered Gene Therapy: This Viral Vector Manufacturing Method Could Widen Its Applicability. BioProcess International 2016, 11,14
  • Cell-Delivered Entry Inhibitors for HIV-1: CCR5 Downregulation and Blocking Virus/Membrane Fusion in Defending the Host Cell Population

    Symonds, G.; Bartlett, JS.; Kiem, HP.; Tsie, M.; Breton, L. AIDS Patient Care STDS 2016 Dec;30(12):545-550

    HIV-1 infection requires the presence of the CD4 receptor on the target cell surface and a coreceptor, predominantly CC-chemokine receptor 5 (CCR5). It has been shown that individuals who are homozygous for a defective CCR5 gene are protected from HIV-1 infection. A novel self-inactivating lentiviral vector LVsh5/C46 (Cal-1) has been engineered to block HIV-1 infection with two viral entry inhibitors, conferring resistance to HIV-1 infection from both CCR5 and CXCR4 tropic strains. Cal-1 encodes a short hairpin RNA (sh5) to downregulate CCR5 and C46, an HIV-1 fusion inhibitor. Gene therapy by Cal-1 is aimed at transducing CD4+ T cells and CD34+ hematopoietic stem/progenitor cells in an autologous transplant setting. Pre-clinical safety and efficacy studies in vitro and in vivo (humanized mouse model and nonhuman primates) have shown that Cal-1 is safe with no indication of any toxicity risk and acts to decrease viral load and increase CD4 counts. Two clinical trials are underway using Cal-1: a phase I/II study to assess safety and feasibility in an adult HIV-1-positive population not on antiretroviral therapy (ART); and a second Fred Hutchinson Investigator Initiated phase I study to assess safety and feasibility in adults with HIV-1-associated non-Hodgkin or Hodgkin lymphoma.

  • Multilineage Polyclonal Engraftment of Cal-1 Gene-Modified Cells and In Vivo Selection After SHIV Infection in a Nonhuman Primate Model of AIDS.

    Peterson, C.W.; Haworth, K.G.; Burke, B.P.; Polacino, P.; Norman, K.K.; Adair, J.E.; Hu, S.-L.; Bartlett, J.S.; Symonds, G.P.; Kiem, H.-P. Multilineage polyclonal engraftment of Cal-1 gene modified cells and in vivo selection after SHIV infection in a nonhuman primate model of AIDS. Molecular Therapy - Methods & Clinical Development 2016, 3, 16.

    We have focused on gene therapy approaches to induce functional cure/remission of HIV-1 infection. Here, we evaluated the safety and efficacy of the clinical grade anti-HIV lentiviral vector, Cal-1, in pigtailed macaques (Macaca nemestrina). Cal-1 animals exhibit robust levels of gene marking in myeloid and lymphoid lineages without measurable adverse events, suggesting that Cal-1 transduction and autologous transplantation of hematopoietic stem cells are safe, and lead to long-term, multilineage engraftment following myeloablative conditioning. Ex vivo, CD4+ cells from transplanted animals undergo positive selection in the presence of simian/human immunodeficiency virus (SHIV). In vivo, Cal-1 gene-marked cells are evident in the peripheral blood and in HIV- relevant tissue sites such as the gastrointestinal tract. Positive selection for gene-marked cells is observed in blood and tissues following SHIV challenge, leading to maintenance of peripheral blood CD4+ T-cell counts in a normal range. Analysis of Cal-1 lentivirus integration sites confirms polyclonal engraftment of gene-marked cells. Following infection, a polyclonal, SHIV-resistant clonal repertoire is established. These findings offer strong preclinical evidence for safety and efficacy of Cal-1, present a new method for tracking protected cells over the course of virus-mediated selective pressure in vivo, and reveal previously unobserved dynamics of virus-dependent T-cell selection.

  • CCR5 Targeted Cell Therapy for HIV and Prevention of Viral Escape

    Hütter, G.; Bodor, J.; Ledger, S.; Boyd, M.; Millington, M.; Tsie, M.; Symonds, G. CCR5 Targeted Cell Therapy for HIV and Prevention of Viral Escape. Viruses 2015, 7, 4.

    Allogeneic transplantation with CCR5-delta 32 (CCR5-d32) homozygous stem cells in an HIV infected individual in 2008, led to a sustained virus control and probably eradication of HIV. Since then there has been a high degree of interest to translate this approach to a wider population. There are two cellular ways to do this. The first one is to use a CCR5 negative cell source e.g., hematopoietic stem cells (HSC) to copy the initial finding. However, a recent case of a second allogeneic transplantation with CCR5-d32 homozygous stem cells suffered from viral escape of CXCR4 quasi-species. The second way is to knock down CCR5 expression by gene therapy. Currently, there are five promising techniques, three of which are presently being tested clinically. These techniques include zinc finger nucleases (ZFN), clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 nuclease (CRISPR/Cas9), transcription activator-like effectors nuclease (TALEN), short hairpin RNA (shRNA), and a ribozyme. While there are multiple gene therapy strategies being tested, in this review we reflect on our current knowledge of inhibition of CCR5 specifically and whether this approach allows for consequent viral escape.

  • Engineering HIV-1-Resistant T-Cells from Short-Hairpin RNA-Expressing Hematopoietic Stem/Progenitor Cells in Humanized BLT Mice

    Ringpis, G.E.; Shimizu, S.; Arokium, H.; Camba-Colón, J.; Carroll, M.V.; Cortado, R.; Xie, Y.; Kim, P.Y.; Sahakyan, A.; Lowe, E.L.; Narukawa, M.; Kandarian, F.N.; Burke, B.P.; Symonds, G.P.; An, D.S.; Chen, I.S.; Kamata, M. Engineering HIV-1-Resistant T-Cells from Short-Hairpin RNA-Expressing Hematopoietic Stem/Progenitor Cells in Humanized BLT Mice. PLoS One. 2012, 7, 12.

    Down-regulation of the HIV-1 coreceptor CCR5 holds significant potential for long-term protection against HIV-1 in patients. Using the humanized bone marrow/liver/thymus (hu-BLT) mouse model which allows investigation of human hematopoietic stem/progenitor cell (HSPC) transplant and immune system reconstitution as well as HIV-1 infection, we previously demonstrated stable inhibition of CCR5 expression in systemic lymphoid tissues via transplantation of HSPCs genetically modified by lentiviral vector transduction to express short hairpin RNA (shRNA). However, CCR5 down-regulation will not be effective against existing CXCR4-tropic HIV-1 and emergence of resistant viral strains. As such, combination approaches targeting additional steps in the virus lifecycle are required. We screened a panel of previously published shRNAs targeting highly conserved regions and identified a potent shRNA targeting the R-region of the HIV-1 long terminal repeat (LTR). Here, we report that human CD4(+) T-cells derived from transplanted HSPC engineered to co-express shRNAs targeting CCR5 and HIV-1 LTR are resistant to CCR5- and CXCR4- tropic HIV-1-mediated depletion in vivo. Transduction with the combination vector suppressed CXCR4- and CCR5- tropic viral replication in cell lines and peripheral blood mononuclear cells in vitro. No obvious cytotoxicity or interferon response was observed. Transplantation of combination vector-transduced HSPC into hu-BLT mice resulted in efficient engraftment and subsequent stable gene marking and CCR5 down-regulation in human CD4(+) T-cells within peripheral blood and systemic lymphoid tissues, including gut-associated lymphoid tissue, a major site of robust viral replication, for over twelve weeks. CXCR4- and CCR5- tropic HIV-1 infection was effectively inhibited in hu-BLT mouse spleen-derived human CD4(+) T-cells ex vivo. Furthermore, levels of gene-marked CD4(+) T-cells in peripheral blood increased despite systemic infection with either CXCR4- or CCR5- tropic HIV-1 in vivo. These results demonstrate that transplantation of HSPCs engineered with our combination shRNA vector may be a potential therapy against HIV disease.

  • Engineering Cellular Resistance to HIV-1 Infection In Vivo Using a Dual Therapeutic Lentiviral Vector

    Burke, B.P.; Levin, B.R.; Zhang, J.; Sahakyan, A.; Boyer, J.; Carroll, M.V.; Colón, J.C.; Keech, N.; Rezek, V.; Bristol, G.; Eggers, E.; Cortado, R.; Boyd, M.P.; Impey, H.; Shimizu, S.; Lowe, E.L.; Ringpis, G.E.; Kim, S.G.; Vatakis, D.N.; Breton, L.R.; Bartlett, J.S.; Chen, I.S.; Kitchen, S.G.; An, D.S.; Symonds, G.P. Engineering Cellular Resistance to HIV-1 Infection In Vivo Using a Dual Therapeutic Lentiviral Vector. Molecular Therapy—Nucleic Acids, 2015, 4, 23.

    We described earlier a dual-combination anti-HIV type 1 (HIV-1) lentiviral vector (LVsh5/C46) that downregulates CCR5 expression of transduced cells via RNAi and inhibits HIV-1 fusion via cell surface expression of cell membrane-anchored C46 antiviral peptide. This combinatorial approach has two points of inhibition for R5-tropic HIV-1 and is also active against X4-tropic HIV-1. Here, we utilize the humanized bone marrow, liver, thymus (BLT) mouse model to characterize the in vivo efficacy of LVsh5/C46 (Cal-1) vector to engineer cellular resistance to HIV-1 pathogenesis. Human CD34+ hematopoietic stem/progenitor cells (HSPC) either nonmodified or transduced with LVsh5/C46 vector were transplanted to generate control and treatment groups, respectively. Control and experimental groups displayed similar engraftment and multilineage hematopoietic differentiation that included robust CD4+ T-cell development. Splenocytes isolated from the treatment group were resistant to both R5- and X4-tropic HIV-1 during ex vivo challenge experiments. Treatment group animals challenged with R5-tropic HIV-1 displayed significant protection of CD4+ T-cells and reduced viral load within peripheral blood and lymphoid tissues up to 14 weeks postinfection. Gene-marking and transgene expression were confirmed stable at 26 weeks post-transplantation. These data strongly support the use of LVsh5/C46 lentiviral vector in gene and cell therapeutic applications for inhibition of HIV-1 infection.

  • CCR5 as a Natural and Modulated Target for Inhibition of HIV

    Burke, B.P.; Boyd, M.P.; Impey , H.; Breton, L.R.; Bartlett, J.S.; Symonds, G. P.; Hütter, G. CCR5 as a Natural and Modulated Target for Inhibition of HIV. Viruses, 2014, 6(1), 54-68.

    Human immunodeficiency virus type 1 (HIV-1) infection of target cells requires CD4 and a co-receptor, predominantly the chemokine receptor CCR5. CCR5-delta32 homozygosity results in a truncated protein providing natural protection against HIV infection—this without detrimental effects to the host—and transplantation of CCR5-delta32 stem cells in a patient with HIV (“Berlin patient”) achieved viral eradication. As a more feasible approach gene-modification strategies are being developed to engineer cellular resistance to HIV using autologous cells. We have developed a dual therapeutic anti-HIV lentiviral vector (LVsh5/C46) that down-regulates CCR5 and inhibits HIV-1 fusion via cell surface expression of the gp41-derived peptide, C46. This construct, effective against multiple strains of both R5- and X4-tropic HIV-1, is being tested in Phase I/II trials by engineering HIV-resistant hematopoietic cells.

  • Preclinical safety and efficacy of an anti–HIV-1 lentiviral vector containing a short hairpin RNA to CCR5 and the C46 fusion inhibitor

    Wolstein, O.; Boyd, M.; Millington, M.; Impey, H.; Boyer, J.; Howe, A.; Delebecque, F.; Cornetta, K.; Rothe, M.; Baum, C.; Nicolson, T.; Koldej, R.; Zhang, J.; Keech, N.; Camba-Colon, J.; Breton, L.; Bartlett, J.; An, D.S.; Chen, I.S.Y.; Burke, B.; Symonds, G.P. Preclinical safety and efficacy of an anti–HIV-1 lentiviral vector containing a short hairpin RNA to CCR5 and the C46 fusion inhibitor. Molecular Therapy — Methods & Clinical Development 2014, 1, 11.

    Gene transfer has therapeutic potential for treating HIV-1 infection by generating cells that are resistant to the virus. We have engineered a novel self-inactivating lentiviral vector, LVsh5/C46, using two viral-entry inhibitors to block early steps of HIV-1 cycle. The LVsh5/C46 vector encodes a short hairpin RNA (shRNA) for downregulation of CCR5, in combination with the HIV-1 fusion inhibitor, C46. We demonstrate here the effective delivery of LVsh5/C46 to human T cell lines, peripheral blood mononuclear cells, primary CD4+ T lymphocytes, and CD34+ hematopoietic stem/progenitor cells (HSPC). CCR5-targeted shRNA (sh5) and C46 peptide were stably expressed in the target cells and were able to effectively protect gene-modified cells against infection with CCR5- and CXCR4-tropic strains of HIV-1. LVsh5/C46 treatment was nontoxic as assessed by cell growth and viability, was noninflammatory, and had no adverse effect on HSPC differentiation. LVsh5/C46 could be produced at a scale sufficient for clinical development and resulted in active viral particles with very low mutagenic potential and the absence of replication-competent lentivirus. Based on these in vitro results, plus additional in vivo safety and efficacy data, LVsh5/C46 is now being tested in a phase 1/2 clinical trial for the treatment of HIV-1 disease.

  • The use of cell-delivered gene therapy for the treatment of HIV/AIDS

    Symonds, G.P.; Johnstone, H.A.; Millington, M.L.; Boyd, M.P.; Burke, B.P.; Breton, L.R. The use of cell-delivered gene therapy for the treatment of HIV/AIDS. Immunol Res., 2010 Dec; 48(1-3): 84-98>

    HIV/AIDS is a disease that impairs immune function, primarily by decreasing T-lymphocyte count. Its progression can be contained by highly active antiretroviral therapy (HAART), but there are side effects that can be severe, and the development of resistance often forces the physician to modify the HAART regimen. There are no vaccines available for HIV. An alternative approach that could provide a path to a curative therapy is the use of cell-delivered gene therapy in which an anti-HIV gene(s) is introduced into hematopoietic cells to produce a population that is protected from the effects of HIV. In this paper, we review the field and discuss an approach using a short hairpin RNA to CCR5, an important co-receptor for HIV.

  • Gene Therapy. Intracellular Immunization

    Baltimore, D. Gene therapy. Intracellular immunization. Nature, 335, 395-396 (29 September 1988).