Proceedings of the first academic symposium on developing, qualifying and operating a cell and gene therapy manufacturing facility.

A significant portion of the more than 1000 candidate cell and gene therapy products currently under clinical investigation (clinicaltrials.gov) are born out of academic research centers affiliated with universities, hospitals and non-profit research institutions. Supporting these efforts are myriad academic clinical materials production facilities with more than 40 such facilities currently operational in the United States alone. In March 2018, Stanford University's Laboratory for Cell and Gene Therapy held a symposium with the leaders and staff of more than 25 similar facilities to discuss the collective experience in developing, qualifying and operating cell and gene therapy manufacturing facilities according to current Good Manufacturing Practices. Topics included facility design, construction, staffing and operations and compliance. Leaders from several institutions gave overviews of the history of development of the facilities and discussed challenges and opportunities they had experienced over the past 10-20 years of operations. Working sessions were also held to discuss specific aspects of Process Development, Manufacturing, Quality Systems, Regulatory Affairs and Business Development with all participants contributing to the discussions. We summarize here the findings of this inaugural meeting with an emphasis on best practices and suggested guidelines for operations.

Genomic editing of the HIV-1 coreceptor CCR5 in adult hematopoietic stem and progenitor cells using zinc finger nucleases.

The HIV-1 coreceptor CCR5 is a validated target for HIV/AIDS therapy. The apparent elimination of HIV-1 in a patient treated with an allogeneic stem cell transplant homozygous for a naturally occurring CCR5 deletion mutation (CCR5(Δ32/Δ32)) supports the concept that a single dose of HIV-resistant hematopoietic stem cells can provide disease protection. Given the low frequency of naturally occurring CCR5(Δ32/Δ32) donors, we reasoned that engineered autologous CD34(+) hematopoietic stem/progenitor cells (HSPCs) could be used for AIDS therapy. We evaluated disruption of CCR5 gene expression in HSPCs isolated from granulocyte colony-stimulating factor (CSF)-mobilized adult blood using a recombinant adenoviral vector encoding a CCR5-specific pair of zinc finger nucleases (CCR5-ZFN). Our results demonstrate that CCR5-ZFN RNA and protein expression from the adenoviral vector is enhanced by pretreatment of HSPC with protein kinase C (PKC) activators resulting in >25% CCR5 gene disruption and that activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway is responsible for this activity. Importantly, using an optimized dose of PKC activator and adenoviral vector we could generate CCR5-modified HSPCs which engraft in a humanized mouse model (albeit at a reduced level) and support multilineage differentiation in vitro and in vivo. Together, these data establish the basis for improved approaches exploiting adenoviral vector delivery in the modification of HSPCs.

Engraftment and lineage potential of adult hematopoietic stem and progenitor cells is compromised following short-term culture in the presence of an aryl hydrocarbon receptor antagonist.

Hematopoietic stem cell gene therapy for HIV/AIDS is a promising alternative to lifelong antiretroviral therapy. One of the limitations of this approach is the number and quality of stem cells available for transplant following in vitro manipulations associated with stem cell isolation and genetic modification. The development of methods to increase the number of autologous, gene-modified stem cells available for transplantation would overcome this barrier. Hematopoietic stem and progenitor cells (HSPC) from adult growth factor-mobilized peripheral blood were cultured in the presence of an aryl hydrocarbon receptor antagonist (AhRA) previously shown to expand HSPC from umbilical cord blood. Qualitative and quantitative assessment of the hematopoietic potential of minimally cultured (MC-HSPC) or expanded HSPC (Exp-HSPC) was performed using an immunodeficient mouse model of transplantation. Our results demonstrate robust, multilineage engraftment of both MC-HSPC and Exp-HSPC although estimates of expansion based on stem cell phenotype were not supported by a corresponding increase in in vivo engrafting units. Bone marrow of animals transplanted with either MC-HSPC or Exp-HSPC contained secondary engrafting cells verifying the presence of primitive stem cells in both populations. However, the frequency of in vivo engrafting units among the more primitive CD34+/CD90+ HSPC population was significantly lower in Exp-HSPC compared with MC-HSPC. Exp-HSPC also produced fewer lymphoid progeny and more myeloid progeny than MC-HSPC. These results reveal that in vitro culture of adult HSPC in AhRA maintains but does not increase the number of in vivo engrafting cells and that HSPC expanded in vitro contain defects in lymphopoiesis as assessed in this model system. Further investigation is required before implementation of this approach in the clinical setting.

Enhanced genetic modification of adult growth factor mobilized peripheral blood hematopoietic stem and progenitor cells with rapamycin.

Genetic modification of adult human hematopoietic stem and progenitor cells (HSPCs) with lentiviral vectors leads to long-term gene expression in the progeny of the HSPCs and has been used to successfully treat several monogenic diseases. In some cases, the gene-modified cells have a selective growth advantage over nonmodified cells and eventually are the dominant engrafted population. However, in disease indications for which the gene-modified cells do not have a selective advantage, optimizing transduction of HSPC is paramount to successful stem cell-based gene therapy. We demonstrate here that transduction of adult CD34+ HSPCs with lentiviral vectors in the presence of rapamycin, a widely used mTORC1 inhibitor, results in an approximately threefold increase in stable gene marking with minimal effects on HSPC growth and differentiation. Using this approach, we have demonstrated that we can enhance the frequency of gene-modified HSPCs that give rise to clonogenic progeny in vitro without excessive increases in the number of vector copies per cell or changes in integration pattern. The genetic marking of HSPCs and expression of transgenes is durable, and transplantation of gene-modified HSPCs into immunodeficient mice results in high levels of gene marking of the lymphoid and myeloid progeny in vivo. The prior safe clinical history of rapamycin in other applications supports the use of this compound to generate gene-modified autologous HSPCs for our HIV gene therapy clinical trials.

Optimized processing of growth factor mobilized peripheral blood CD34+ products by counterflow centrifugal elutriation.

Cell separation by counterflow centrifugal elutriation has been described for the preparation of monocytes for vaccine applications, but its use in other current good manufacturing practice (cGMP) operations has been limited. In this study, growth factor-mobilized peripheral blood progenitor cell products were collected from healthy donors and processed by elutriation using a commercial cell washing device. Fractions were collected for each product as per the manufacturer's instructions or using a modified protocol developed in our laboratory. Each fraction was analyzed for cell count, viability, and blood cell differential. Our data demonstrate that, using standard elutriation procedures, >99% of red blood cells and platelets were removed from apheresis products with high recoveries of total white blood cells and enrichment of CD34+ cells in two of five fractions. With modification of the basic protocol, we were able to collect all of the CD34+ cells in a single fraction. The CD34-enriched fractions were formulated, labeled with a ferromagnetic antibody to CD34, washed using the Elutra device, and transferred directly to a magnetic bead selection device for further purification. CD34+ cell purities from the column were extremely high (98.7 ± 0.9%), and yields were typical for the device (55.7 ± 12.3%). The processes were highly automated and closed from receipt of the apheresis product through formulation of target-enriched cell fractions. Thus, elutriation is a feasible method for the initial manipulations associated with primary blood cell therapy products and supports cGMP and current good tissue practice-compliant cell processing.

Manufacturing of large numbers of patient-specific T cells for adoptive immunotherapy: an approach to improving product safety, composition, and production capacity.

We have developed an innovative system for ex vivo processing of patient-specific cell products to produce large numbers of T-lymphocytes in support of phase 2 adoptive immunotherapy trials for hematologic malignancies. Extensive efforts were undertaken to close the cell processing system to improve the safety profile of the process and comply with new federal regulations regarding cell and tissue processing. Our results demonstrate that apheresis products can be processed in a closed system (Cytomate) with similar yields (approximately 4 x 10(9) mononuclear cells/apheresis) and recoveries (approximately 60% of starting mononuclear cells) to manual cell processing. Cells processed with this system could be cryopreserved for up to 5 months without significant loss of recovery or viability. Additionally, we have evaluated the use of gas permeable bags and developed perfusion bioreactor protocols in which T cells can be rapidly produced in excess of 10(10) viable cells per liter of culture. Using similar methods for upfront processing, we have also developed methods for positive selection and ex vivo culture of CD4+ T cells that result in 200 to 800-fold expansion of fresh or cryopreserved samples. T cells produced in these systems were shown to retain activation-induced cytolytic capability and TH1/TH2 cytokine production as a measure of biologic potency. These new methods allow for more efficient production multiple patient-specific products by satisfying the basic tenants of safety and efficacy required for early phase clinical trials of cell products.