GENE THERAPY - Hematopeietic Stem Cell-based, Preclinical Murine Models, Product and Process Development, IND to NDA, PAI, Commercialization, and Launch
MedScope has substantial experience in all aspects of Gene Therapy. Hematopoietic stem cell-based gene therapy has the incredible potential to cure literally all disease having genetic cause, including for example hematologic disorders such as hemophilia or sickle cell disease. Since these diseases have a genetic basis that results in altered or disrupted production of critical proteins, gene therapy can improve or even fully correct these disorders by targeting hematopoietic stem cells to re-populate the body with blood cells containing the corrected gene. In patients that cannot find a suitable bone marrow donor for allogenic bone marrow transplant, gene therapy offers an alternative method that utilizes a patient’s own cells. For this type of therapy, it is not only important to achieve efficient genetic modification, but the stem cell properties must be preserved so that the cells can continue to self-renew. MedScope has the ability to test transduced cells in preclinical murine models, thereby enabling rapid early  preclinical simulation of gene therapy treatments as well as early product and process optimoization. MedScope is a rapid development organization, and in most instances can initiate value-based discussions within 24 hours. Please contact us at 802-236-8650 or

Murine Preclinical Studies
For transduction efficiency studies, murine models are typically selected  between 8-10 weeks, typically without randomization in order to quickly progress early learning and product/process development. Based on statistical power estimates (alpha = 0.05, power = 0.8, sigma = 0.3) assuming fVIII expression levels drawn from previous unpublished animal studies, a minimum of three mice per group would be needed to determine differences. Total animal numbers (n) for each group are given. An initial pilot experiment was conducted in which Sca-1+ cells were transduced for 5 hours in microfluidics and compared to optimized 6-well transductions that had previously been investigated by the Spencer/Doering labs. The optimized protocol called for two doses of LV over a period of 28 hours. Due to the extremely high cell volumetric concentration used in the microfluidic, transduction times were reduced to 5 hours, which has been shown to be an effective length for microfluidic transduction without inducing detrimental effects from nutrient deprivation. Furthermore, extended ex vivo culture of stem cells has been shown to affect differentiation and self renewal capacity of hematopoietic stem cells, and was therefore minimized for our studies. The microfluidic transductions used either the same amount of LV or half as much. This experiment is outlined below in Figure 6.1, and informed the design of our second animal experiment, which aimed to compare and minimize vector dosage between systems for identical transduction times.

Harvest and Culture of Sca-1+ Cells
​Bone marrow was isolated from cleaned hind leg femurs and tibias of C57BL/6 E16 hemophilia A mice with the CD45.1 allele. Sca-1+ cells were incubated with biotin 97 anti-Sca-1 antibody followed by anti-biotin microbeads (Miltenyi Biotec, Inc., San Diego, CA, USA) and passed through a magnetic separation column for positive section. Isolated cells were then cultured overnight at a density of 2x106 cells/mL in StemPro media supplemented with stem cell factor (100 ng/mL), murine interleukin-3 (20 ng/mL), human interleukin-11 (100 ng/mL), human Flt-3 ligand (100 ng/mL), StemPro nutrient supplement (40x), L-glutamine (100x), and penicillin/streptomycin (100x).

​Transduction of Sca-1+ Cells
On the day of transduction, 2 million cells were loaded into each bare microfluidic and 6-well with 8 µg/mL Polybrene as described above with a clinical grade fVIII-LV for 5 hours. Cells were then collected, washed, and re-suspended in fresh PBS for transplant.

Transplantation of Transduced Sca-1+ Cells 
At least four hours prior to transplantation, a separate cohort of 8- to 10-week-old C56BL/6 E16 hemophilia A mice with the CD45.2 allele were given two doses of lethal irradiation at 11 Gy Rad four hours apart. After transduction and washing of LV from the donor cells, 1,000,000 cells were transplanted into each host mouse via retro-orbital injection. 

​Bi-weekly Assessment of Sca-1+ Cell Transduction and Engraftment
​Mice were bled every two weeks for the first 8 weeks via tail vein microsampling. Flow cytometry was run on collected blood cells and stained for CD45.1 and CD45.2 to assess engraftment. The following antibody-fluorophore conjugates were used for flow cytometry: CD45.2-APC (558702), Gr1-APC-Cy7 (557661), Mac1-APC-Cy7 (557657),  98CD45.1-PE (553776), CD3e-V450 (560801), and CD45R/B220-PE-Cy7 (552772) (BD Biosciences, San Jose, CA, USA). Complete blood counts were also conducted to monitor populations of various white blood cells including lymphocytes, monocytes, and granulocytes. Plasma was isolated and used to measure fVIII plasma levels via commercially available chromogenic substrate assay (Chromogenix Coatest SP FVIII, diaPharma, West Chester, OH, USA). Note C56BL/6 E16 hemophilia A mice with the CD45.2 allele were given two doses of lethal irradiation at 11 Gy Rad four hours apart. 

Quantification of Vector Copies in Blood, Spleen, and Bone Marrow 
After 16 weeks, mice were euthanized, and cells from blood, spleen, and bone marrow were harvested for RT-PCR to quantify LV copy number. Vector copy number analysis were conducted. The operator was blinded to the cell samples before processing for vector copy number.  

Vector Copy Number Harvested Blood Cells
The vector copy number of harvested blood cells showed that vector integration was highest in mice that were transduced with the least amount of LV. Therefore, the VCN utilization efficiency was also highest (Figure 6.4b). These results indicate that biological barriers such as LV binding may have become rate limiting, leading 
to saturation since doubling the amount of LV did not further improve vector integration. Combined with our previous data and basic analytical models of particle diffusion, we expected greater VCN due to the increased LV availability. As such, these data served as an upper limit of LV usage in our animal experiments since microfluidics using the same amounts of LV as the 6-wells did not demonstrate increased utilization efficiency, and only served to decrease transduction time. Overall, these data show that microfluidics can achieve sufficient gene transfer while reducing both LV usage and significantly reducing transduction times. 

​Cell viability 
Cell viability can be difficult to assess following in vitro transductions since any cells that were negatively impacted by microfluidic transduction would quickly be selected against while viable cells continued to proliferate. Therefore, in vivo assessment of cell engraftment is critical for determining if microfluidics negatively impacted the cells. Donor mice were selected for the CD45.1 allele while recipient mice had the CD45.2 allele. Due to lethal irradiation of recipient mice prior to transplantation, nearly all CD45.2+ cells were eliminated. Therefore, all blood cells originating from the transplanted HSCs would be CD45.1+ while poor engraftment would result in an increase in CD45.2+ cells.
Monitoring of white blood cell counts, particularly granulocyte, monocyte, and lymphocyte counts, were important to assess re-establishment of the immune system. Furthermore, due to the CD68-specific promoter used in the fVIII-LV, transgene expression would be directed toward cells of the myeloid lineage, which include granulocytes and monocytes. Flow cytometry showed that engraftment was high in all animals, and increased steadily over time, stabilizing after 8 weeks. Other than a single outlier in each microfluidic group, all mice displayed >90% CD45.1+ cells and <10% CD45.2+ cells. 

Viral Vector Integration
VCN utilization efficiency was calculated from the various tissues harvested and assessed for copy number (Figure 6.8a). Individual VCN for blood (Figure 6.8b), bone marrow (Figure 6.8c), and spleen (Figure 6.8). Comparable VCN utilization efficiency was achieved in both (1x) and (0.25x) microfluidics except for bone marrow cells. Though 
only blood VCN utilization efficiency was statistically significant compared to the 6-well transductions (p<0.05), the majority of mice receiving cells transduced in microfluidics demonstrated greater utilization efficiency. Despite significantly lower vector dosages and exception of bone marrow VCN. Individual VCN and average fVIII values for each mouse are given below in Table 6.2. 

From these studies, we were able to definitively show that the microfluidic transduction platform did not have any negative impacts on cell viability, engraftment, or these mice were not compromised as demonstrated by two separate cohorts which survived to 16 and 19 weeks post-transplant. All mice transplanted with microfluidic transduced of fVIII. Using even less vector in the direct 6-well comparisons, three of five mice were Although the transduction times were already significantly shortened compared to clinical protocols, assessment of transduction kinetics with these cells would be interesting to determine the minimum time required for effective transduction.

Future Opportunities In Process/Product Development of GT Using Murine Models
translated state-of-the-art transduction protocols to a microfluidic platform enabling significant reduction in LV requirements by leveraging the  micron-scale heights of microfluidics to overcome diffusion limitations of current systems.  Moreover, the high surface area-to-volume ratio of microfluidics efficiently brings LV into cell contact before degradation occurs, reducing transduction times while minimizing LV waste and total amounts used by enabling large quantities of cells to be exposed to sufficient vector concentrations. Transduction efficiency can be further maximized by incorporating existing transduction-enhancing methods. Overall, the microfluidic platform outperforms current clinical transduction platforms in all aspects studied.  

As shown from our primary cell data, diffusion limitations are not the only barriers to overcome in developing a novel microfluidic platform for gene therapy. While our system currently enables enhanced transduction simply from adapting existing protocols combined with leveraging the increased mass transport afforded by microfluidics, there are still many other areas where improvements can be targeted by changing the design or incorporating new microfluidic features that may also be used to actively enhance vectorcell interactions. Based on the insights gained from this work, it may be equally advantageous to leverage the microfluidic system to reduce the expensive cytokine 
requirements currently needed to effectively stimulate or activate primary cells for transduction. Future work can focus on using our microfluidics for short term cell culture or stimulation to build toward an all-in-one system that can be compatible with existing apheresis machines.  

Among potential device improvements and implementations, additional efforts need to be put forth in further characterizing primary T cell and hematopoietic stem cell microfluidic transduction. One potential route is to assess CAR-LVs targeting CD19 for acute lymphoblastic leukemia. Our previous T cell work was only able to assess vector 
integration as a primary output due to limitations in LV availability. However, it should be a top priority to transduce T cells with a CAR-LV so that functional assays can be conducted for efficacy or safety profiling. With T cells, tumor cell-specific cytotoxicity could be measured to quantify the anti-cancer potential of microfluidic-generated CAR T 
cells. Future hematopoietic stem cell work should investigate the efficacy of microfluidics in transducing human CD34+ cells, which are an even more difficult target for genetic modification. The cells could then be assessed with the human colony forming cell assay using methylcellulose-based media to determine if engraftment has been compromised. Alternatively, transduced human CD34+ cells could be transplanted into humanized NOD SCID mice to assess engraftment potential.  

Finally, continued efforts to scale up are necessary to make a true impact on gene therapy globalization and commercialization. While mock-ups of potential mold designs have been considered as discussed in Section 3.5, future designs do not have to be limited to PDMS-based devices. One potential idea is to take existing cell culture bags and use a hot embossing process to pattern channels within the bag so that the geometry can be constrained to have high surface area to volume ratio. Another thought would be to use gaspermeable sheets of similar materials to cell culture bags and use a xurographic process to pattern channels and bond two layers together to form a channel in a bag again. This design  could eventually intersect with the previously discussed idea of having the device directly  that an order of magnitude reduction in vector costs per patient may be attainable by using 
microfluidics. Since the LV manufacturing process currently induces high toxicity in the mammalian cell required for production, it is not possible to perform long-term continuousimprovements in transduction efficiency will only be enhanced if better technologies arisegreater efficiency. As the current strategy in clinical gene therapy is to apply as much vector onto cells as possible without inducing detrimental toxicity, incorporating microfluidic transduction can significantly reduce the wastes inherent in current processes and overcome issues related to the various sources of variability in vector titration and inconsistent infectivity between various cell types. With clinical gene therapy rapidly 
advancing with definite evidence of success and licensed products, associated advances in vector manufacturing and utilization are going to be essential to routine clinical 
implementation and globalization.  

How To Reach Us
MedScope is a rapid development organization, and in most instances can initiate and address a wide range of value-based discussions within 24 hours. Our teams and wide technological network in the biopharmaceutical industry enables us to quickly bring substantial knowledge and problem solving skills to help you quickly solve problems. For rapid response, please contact us at 802-236-8650 or 

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