Press Reports

The Cellectis news from yesterday and subsequent Sangamo mention has Genomeweb.com placing SGMO is some heady company.

(snip)
Still, the Journal notes that a number of companies like Pfizer, AstraZeneca, Novartis, and Sangamo BioSciences and academic researchers are exploring gene editing, including using zinc finger nucleases and CRISPR.

https://www.genomeweb.com/scan/gene-editing-remission

Seeking Alpha Article that Relates to Sangamo HIV Manufacturing program

http://seekingalpha.com/article/3651256-victory-in-the-car-t-race-may-be-pyrrhic-juno-the-biggest-loser-cellectis-too-late-for-the-spoils?page=2

Summary

"T cell manufacturing is robust and dependable...A handful of academic centers have successfully established manufacturing procedures that have proven to be reproducible and dependable" (Sadelain, 2015).

"The field ... has a dearth of data as to whether centralized manufacturing of patient-derived CAR T cells is broadly feasible and does not result in ruinous costs..." (Cooper, 2015).

"Amateurs talk about tactics, but professionals study logistics" applies to military ops but also to clinical ops being transitioned to commercial, centralized manufacturing.

CEOs who think they can take this brilliant science commercial without a road map through the daunting logistics prove themselves amateurs.

Avoid them all until you at least see a road map, especially the biotechs with largest market cap. Consider small-cap contract manufacturers who demonstrate expertise in logistics.

A victory is pyrrhic when even the victor counts the cost too high, and that is how the crowded race for autologous CAR-T cells could end.
Genetic engineering of CARs into T cells is one of the most complex processes known to modern medicine. Central as opposed to point-of-care manufacturing of CAR-T cells for autologous transfusion adds logistical complexity and treatment risk to an intense therapy that already carries great risk to a vulnerable patient group, those with cancer refractory to conventional therapies.
Experts in commercial scale manufacturing warn against the scaling up of immune cell production without a transition period of close collaboration between manufacturer and the academic facilities in which patient-specific, genetically engineered T cell therapies originate: the science behind CAR-T is brilliant but scaling up central manufacturing requires a vastly different skill set (see Preti, 2015 and O'Donnell, 2015).

No one should know more about failed cell manufacturing models than Juno (NASDAQ:JUNO) CEO Hans Bishop who was EVP and COO at Dendreon (OTCPK:DNDNQ) from January 2010 to September 2011. Dendreon attempted to manufacture its own cellular therapeutic. DNDNQ entered Chapter 11 in November 2014.
Dendreon's PROVENGE® sipuleucel-T is a patient-specific but centrally manufactured immunocellular therapy for autologous transfusion in prostate cancer. Despite a favorable impact on overall survival (Kantoff, 2010), the medical community did not think the benefits of Sipuleucel-T justified the $93,000 price tag, sales were disappointing, production costs high - a Dendreon website video shows why: logistics are a potential nightmare.
Harvesting cells from a patient, shipping them to a manufacturer for immunological manipulation, and sending them back for transfusion into the same (we hope) patient is not only risky business, it is risky medicine: Dendreon had difficulty maintaining quality control amidst the complexities of manufacture and distribution. I am not saying that cancer therapy with CAR-T will be as unimpressive as Provenge has been. T cell therapies are poised to become part of the standard-of-care treatments for patients with cancer (Wang and Rivière, 2015). But manufacturing genetically engineered CAR-T cells is longer (2 weeks) and more complex than for Provenge (not genetically modified and takes 3 days) by an order of magnitude.
In a white paper, titled "Manufacturing Cell Therapies: Development Strategies for Commercial Vision", authors Richard Grant and Brian Hampson, VP of Manufacturing Development and Engineering for Progenitor Cell Therapy ((NASDAQ:CLBS)), offer a fairly detailed road map which I have summarized:

1. Client (e.g. Juno) creates a Quality Target Product Profile for future labeling that follows FDA guidelines,
2. Long-term collaboration begins with manufacturing system developer (e.g. PCT/CLBS) visiting client to observe processes currently in use
3. Client formally communicates to manufacturer expectations for numbers of patients to be treated, costs of products, handling and shipping of products, and other requirements
4. client gains a clearer strategic vision to communicate to investors and others
5. CMO (e.g. Invetech/CLBS) builds prototype for small-scale production, produces cells for quality testing by client

I searched JUNO's latest 10K, 10Q and earnings conference calls for evidence that Hans Bishop had some learned some important lessons from the DNDNQ failure, and communicated to investors (as in step 4 above) his road map for success. This is what I do see:

"We've transitioned from an academic process to a commercially scalable one."

How?

"The company entered into a lease for an approximately 68,000 square foot manufacturing facility in Bothell, Washington, which lease commenced in March 2015."

Already?

"We plan to complement the use of one or more CMOs by establishing our own cGMP manufacturing facility to be brought on-line after the first CMO. As described...we have entered into a ten-year lease for a facility that we plan to remodel to support our clinical and commercial manufacturing activities."

Headlong into uncharted waters is what that sounds like to me. Meanwhile, Novartis has already moved into the NJ cell manufacturing plant that Mr. Bishop equipped, and his successor at DNDNQ had to liquidate.
Novartis is too big a company for me to study and present, but I took note of its collaboration with U Penn when the CART19 story made big news in NEJM (Porter, 2011). NVS surely has the liquidity to stay in this CAR-T race longer than JUNO could stay solvent without the aid of Celgene (NASDAQ:CELG). And what an advantage having a ready-made (courtesy of Hans Bishop's Dendreon) cell manufacturing plant when all JUNO has to show its investors is a lease agreement!
David Chang, Kite's (NASDAQ:KITE) chief medical officer, told a Reuters reporter that Kite will minimize overhead and manufacturing costs by building individual modules that can each handle a patient's cells and then will then "build out" with more factory modules as demand increases. That sounds like a good idea, and I think Kite's plan for a commercial manufacturing facility adjacent to Los Angeles International Airport also shows they are logistically minded. Meanwhile, KITE will leave responsibility for cell manufacture to Progenitor Cell Therapy, a subsidiary of Caladrius Biosciences. CLBS bioprocesses and manufactures but does not genetically engineer cells. CLBS operating expenses exclusive of R&D rose faster than revenues in FY 2013 and 2014, and its own cell therapy programs are not doing well. President of this company, Dr. Robert Preti, has posted an article in DDNews, September 2015, provocatively entitled "Building a problem or a solution?" Dr. Preti articulates concerns about the state of his industry and the direction it must go. KITE's dependence on this tiny company with only $21M in PP&E is of some concern - CLBS will need capital to build capacity, and that will be more difficult with its stock price way down this year. Perhaps CLBS will be acquired.
There are some sub-$1B market cap companies that are engineering features into T cells to make them safer and more effective, but for whom logistics is not yet a critical concern.
Bellicum (NASDAQ:BLCM) has a variety of T lymphocyte and other immune cell platforms. Its most advanced product in phases 1 and 2 is BPX-501, an allogeneic, partially matched T cell removed from donor bone marrow prior to transplantation, engineered with a suicide switch, then used to reconstitute the immune system of the recipient of the transplant. Genetically adding suicide switches (Stasi, 2011; Zhou, 2014) allow for these cells to be shut down if they attack the patients own healthy tissues. Bellicum does research in partnership with scientists at Baylor. BPX-201 is a preclinical cancer vaccine that uses a prostate cancer patient's own dendritic white cells for autologous infusion after genetic engineering of a suicide switch and expression of a prostate cancer antigen that will stimulate and immune attack on the cancer. BPX-601 and -401 are preclinical CAR-T cells with suicide switches. Both are for autologous transfusion, so centralized manufacturing for wide distribution will face the same logistical issues.
Adaptimmune (NASDAQ:ADAP) plans to build its manufacturing plant in The Navy Yard in Philadelphia for use in 2016. Ships are known to move slowly, so logistics was probably not a consideration in selecting that location. The ADAP platform is entirely autologous and not off-the-shelf. ADAP has 8 phase 1 or 1/2 trials registered at ClincialTrials.gov, all using T cells with genetically engineered TCR, not CARs. One of these trials which uses anti-NY-ESO-1 TCR for synovial sarcoma is in collaboration NCI. GlaxoSmithKline (NYSE:GSK) is not named as co-sponsor on any of the registered trials but has options on the T cells with TCR engineered against NY-ESO. ADAP form 10-K indicates (page 56) that current CMO is Progenitor Cell Therapy.
Pity that Cellectis (NASDAQ:CLLS) (OTCPK:CMVLF), the only company developing a universal, off-the shelf CAR T product that would be logistically simpler to manufacture and distribute, has registered nary a single clinical T cell trial among the numerous already registered at ClinicalTrials.gov (see Table below). Not only that, but in reviewing a large amount of the literature on the subject, I came across not a single paper authored by Cellectis scientists, and no mention of them by peers in the field. When I first discovered Cellectis in a review of the 2014 ASH abstracts, I thought I had discovered a gem - and it may indeed be a gem. But Cellectis promised a clinical trial in 2015 which as of November has not started or been further discussed. Lead product is another anti-CD19 CAR, but the race against CD19-bearing B-cell malignancies is getting crowded, and Cellectis may have difficulty finding a market by the time its CRO has gotten UCART19 through trials and approved. The "barrier to entry" into the universal donor CAR-T race is not high: it requires that the lymphocytes native TCR be knocked out, and that technology is currently being widely adopted by others.
Conclusions.

• Central bioprocessing and manufacturing of genetically engineered T cells for autologous transfusion is an unproven business model. Those who try it should learn from Dendreon's failure with a much simpler, genetically un-modified cell therapy product.
• Even industry experts admit (Kaiser, 2015):

"It is conceivable to see a future where gene-modified T cells are manufactured at the point-of-care in a facility in close proximity, associated with, or at the hospital."

...that's quite an understatement

• The cost of manufacturing an off-the-shelf, allogeneic rather than autologous, cancer-fighting T cell will be less ruinous.
• Beyond the economic fundamentals, identification the most important and safe molecular targets, and the ability to edit T cell genes translate competitive edge (June, 2015).

Recommendations:

• $5B JUNO is a STRONG SELL
• $3B KITE is a SELL
• $0.9B Cellectis is very late to enter the race particularly for a CD19 CAR-T, but has a platform for allogeneic use. Speculative HOLD.
• $0.8B market cap Adaptimmune, new to the market in May, was noticed by another SA author in September and is worth following.
• $0.3B BLCM is worth watching
• $68 Million market microcap CLBS is also worth watching. CLBS does have the skill set for manufacturing, and its stock will soar if BLCM or ADAP go commercial before they are fully cGMP-capable.

Sangamo BioSciences To Present Data From ZFP Therapeutic Programs At Annual Meeting Of The American Society Of Hematology

Five Presentations Include Non-Human Primate Data from Sangamo's In Vivo Protein Replacement Platform™ Approach for Hemophilia B, and Data from ZFN-Mediated Genome Editing Approach for Hemoglobinopathies

RICHMOND, Calif., Nov. 5, 2015 /PRNewswire/ -- Sangamo BioSciences, Inc. (Nasdaq: SGMO), a leader in therapeutic genome editing, announced today that non-human primate data from its proprietary In Vivo Protein Replacement Platform (IVPRP™) program for hemophilia B, and data from its ZFP Therapeutic^® hemoglobinopathy programs in collaboration with Biogen, will be presented at the 57th Annual Meeting of the American Society of Hematology (ASH). The 2015 ASH meeting will be held in Orlando, FL from December 5-8, 2015.

"Our presentations at this year's ASH meeting highlight the breadth of our highly specific genome editing platform in both in vivo and ex vivo therapeutic applications," said Edward Lanphier, Sangamo's president and chief executive officer. "Data from non-human primates generated in our Factor IX program for hemophilia B demonstrate the potential of our IVPRP strategy to produce clinically beneficial levels of therapeutic protein from a single treatment. We, and our collaborators at Biogen, will also present data from our hemoglobinopathy programs in beta-thalassemia and sickle cell disease, which use an efficient ZFN-mediated knockout approach in hematopoietic stem cells to elevate functional globin to provide a potentially life-long therapeutic effect." 
Sangamo's hemophilia B program is the first therapeutic application of its IVPRP strategy, an in vivo targeted integration strategy that can be leveraged across multiple monogenic diseases that are currently treated using protein or enzyme replacement therapy. Sangamo remains on track to file Investigational New Drug (IND) applications for hemophilia B (Factor IX) and Hurler syndrome (MPS I) by the end of 2015, and several more IND applications, including hemophilia A, Hunter syndrome (MPS II), Gaucher disease and other lysosomal storage disorders in 2016.
Sangamo is collaborating with Biogen to develop a ZFP Therapeutic approach to beta-thalassemia and sickle cell disease (SCD) that replaces deficient expression of the mutant, disease-causing form of beta-globin with expression of functional fetal globin. The companies expect to file IND applications for beta-thalassemia in the first half of 2016 and for SCD in the second half of 2016.
The following presentations are scheduled at the ASH Meeting sessions:
IVPRP

• ZFN-Mediated Gene Targeting at the Albumin Locus in Liver Results in Therapeutic Levels of Human FIX in Mice and Non-Human Primates - Abstract #200
  Session: 801. Gene Therapy and Transfer: Gene Therapy for Hemoglobinopathies and Inherited Bleeding DisordersOral Presentation - Sunday, December 6, 2015: 7:45 AM
  Presenter - Michael C. Holmes, Ph.D., Sangamo BioSciences

Hemoglobinopathies

• Genome Editing of the Bcl11A Erythroid Specific Enhancer in Bone Marrow Derived Hematopoietic Stem and Progenitor Cells for the Treatment of Sickle Cell Disease - Abstract #203
  Session: 801. Gene Therapy and Transfer: Gene Therapy for Hemoglobinopathies and Inherited Bleeding DisordersOral Presentation - Sunday, December 6, 2015: 8:30 AM
  Presenter - Siyuan Tan, Ph.D., Biogen
• Clinical-Scale Genome Editing of the Human BCL11A Erythroid Enhancer for Treatment of the Hemoglobinopathies - Abstract #204
  Session: 801. Gene Therapy and Transfer: Gene Therapy for Hemoglobinopathies and Inherited Bleeding DisordersOral Presentation - Sunday, December 6, 2015: 8:45 AM
  Presenter - Fyodor, D. Urnov, Ph.D., Sangamo BioSciences
• Clonal Analysis of Human Bone Marrow CD34+ Cells Edited by BCL11A-Targeting ZFNs Reveals Clinically Relevant Levels of Gamma Globin Expression in Edited Erythroid Cells - Abstract #3234Session: 801. Gene Therapy and Transfer: Poster IIPoster Session - Sunday, December 6, 2015: 6:00 - 8:00 PM
  Presenter - Kai-Hsin Chang, Ph.D., Biogen

Other Applications

• In Vivo Genome Editing in Neonatal Mouse Liver Preferentially Utilizes Homology Directed Repair - Abstract #4422Session: 801. Gene Therapy and Transfer: Poster IIIPoster Session - Monday, December 7, 2015: 6:00 - 8:00 PM
  Presenter - Xavier M. Anguela, Ph.D., Children's Hospital of Philadelphia

All abstracts for the ASH meeting are available online at 2015 ASH Annual Meeting Abstracts.
About Sangamo's IVPRPThe IVPRP approach makes use of the albumin gene locus, a highly expressing and liver-specific genomic "safe-harbor site", that can be edited with zinc finger nucleases (ZFNs) to accept and express any therapeutic gene. The platform enables the patient's liver to permanently produce therapeutic levels of a corrective protein product such as factor VIII or IX to treat hemophilia, or replacement enzymes to treat lysosomal storage disorders. With such a large capacity for protein production (approximately 15g/day of albumin), which is in excess of the body's requirements, targeting and co-opting only a very small percentage of the albumin gene's capacity is sufficient to produce the needed replacement protein at therapeutically relevant levels with no significant effect on albumin production.
About Sangamo's ZFP Therapeutic Approach to HemoglobinopathiesSangamo's proprietary ZFN genome editing technology enables the correction of SCD and beta-thalassemia. Both diseases manifest after birth, when patients switch from producing functional fetal gamma-globin to a mutant form of adult beta-globin, which causes their condition. Naturally occurring increased levels of therapeutic fetal hemoglobin have been shown to reduce the severity of both SCD and beta-thalassemia disorders in adulthood. In hematopoietic stem and progenitor cells (HSPCs), Sangamo's genome editing technology can be used to precisely disrupt a key DNA sequence that acts as a powerful tissue and developmental stage "Enhancer" of BCL11A expression. BCL11A is a key transcriptional regulator of the switch from fetal to adult globin production. Knockout of the Enhancer results in the disruption of that switch leading to elevation of fetal globin and reduction in the expression of adult globin.
A bone marrow transplant (BMT) of HSPCs from a "matched" related donor (allogeneic BMT) is curative for both diseases. However, this therapy is limited by the scarcity of matched donors and the significant risk of graft versus host disease (GvHD) after transplantation of the foreign cells. By performing genome editing in HSPCs that are isolated from and subsequently returned to the same patient, an autologous HSPC transplant, Sangamo's approach eliminates both the need for a matched donor and the risk of acute and chronic GvHD. The ultimate goal of this approach is to develop a one-time, life-long treatment for SCD and beta-thalassemia.
About SangamoSangamo BioSciences, Inc. is focused on Engineering Genetic Cures^TM for monogenic and infectious diseases by deploying its novel DNA-binding protein technology platform in therapeutic genome editing and gene regulation. The Company has a Phase 2 clinical program to evaluate the safety and efficacy of novel ZFP Therapeutics^® for the treatment of HIV/AIDS (SB-728). Sangamo's other therapeutic programs are focused on monogenic and rare diseases. The Company has formed a strategic collaboration with Biogen Inc. for hemoglobinopathies, such as sickle cell disease and beta-thalassemia, and with Shire International GmbH to develop therapeutics for Huntington's disease. It has also established strategic partnerships with companies in non-therapeutic applications of its technology, including Dow AgroSciences and Sigma-Aldrich Corporation. For more information about Sangamo, visit the Company's website at www.sangamo.com.
ZFP Therapeutic^® is a registered trademark of Sangamo BioSciences, Inc.

ASH Abstracts for Sangamo 2015

200 ZFN-Mediated Gene Targeting at the Albumin Locus in Liver Results in Therapeutic Levels of Human FIX in Mice and Non-Human Primates

Hemophilia is an attractive target for gene therapy, since activity levels as low as 1% to 2% of normal are beneficial and levels of ~5% prevent spontaneous bleeding. Our goal was to provide a single treatment that permanently enables hepatic production of therapeutic levels of hFIX activity to decrease or potentially eliminate the need for prophylactic treatment in hemophilia B patients. We performed targeted in vivo genome editing using 1) two zinc finger nucleases (ZFNs) targeting intron 1 of the albumin locus, and 2) a human F9 donor template construct. The ZFNs and donor template are encoded on separate hepatotropic adeno-associated virus serotype 2/6 (AAV2/6) vectors injected intravenously, resulting in targeted insertion of a corrected copy of the hF9 gene into the albumin locus in a proportion of liver hepatocytes. The albumin locus was selected as a “safe harbor” as production of this most abundant plasma protein exceeds 10 g/day, and moderate reductions in those levels are well-tolerated.  These genome edited hepatocytes produce normal hFIX in therapeutic quantities, rather than albumin, driven by the highly active albumin enhancer/promoter, to treat hemophilia B; the genetic modification is expected to be sustained even in the face of hepatocyte turnover, making this approach attractive for treating young children with hemophilia before the appearance of significant organ damage. Transformed and primary human hepatocytes transduced in vitro with AAV2/6 encoding human albumin ZFNs and a promoterless hF9 transgene were shown to secrete hFIX. Extensive molecular analyses demonstrated that this was due to targeted integration of the hF9 transgene at the albumin locus and splicing of this gene into the albumin transcript. By employing AAV2/6 delivery of murine-specific ZFNs in vivo, stable levels of hFIX were observed in blood of mice injected with the albumin ZFNs and hF9 transgene donor. C57BL/6 mice were administered vehicle (n=20) or AAV2/6 vectors (n=25) encoding mouse surrogate reagents at 1.0 x10¹³ vector genome (vg)/kg via tail vein injection. ELISA analysis of plasma hFIX in the treated mice showed peak levels of 50-1053 ng/mL that were sustained for the duration of the 6-month study. Analysis of FIX activity from mouse plasma confirmed bioactivity commensurate with expression levels. Next, we report the feasibility of this approach in non-human primates (NHPs), showing that a single intravenous co-infusion of AAV2/6 vectors encoding the NHP targeted albumin-specific ZFNs and a human F9 donor at 1.2x10¹³ vg/kg (n=5/group) resulted in >50 ng/mL (>1% of normal) in this large animal model. The use of higher AAV2/6 doses (up to 1.5x10¹⁴ vg/kg) yielded plasma hFIX levels up to 1000 ng/ml (or 20% of normal) in several animals and up to 2000 ng/ml (or 50% of normal) in a single animal, for the duration of the study (3 months). The treatment was well tolerated in mice and NHPs, with no significant toxicological findings related to AAV2/6 ZFN + donor treatment in either species at therapeutic doses. Together, these data support a clinical trial to determine if a single co-administration of ZFN and donor AAV vectors is sufficient to enable therapeutic and potentially lifelong production of the clotting factor for the treatment of Hemophilia B.

204 Clinical-Scale Genome Editing of the Human BCL11A Erythroid Enhancer for Treatment of the Hemoglobinopathies

We describe here a fundamentally novel way to develop a disease therapeutic: combining genome-wide association studies (GWAS) with targeted genome editing to create, in a clinically compliant setting, a disease-ameliorating genotype in the patient’s own cells. In β-thalassemia, elevated levels of fetal hemoglobin (HbF) lessen or eliminate disease symptoms, thus making a reversal of HbF silencing in patients an appealing therapeutic strategy. Loss-of-function variants in the erythroid-specific enhancer of the fetal globin repressor, BCL11A, elevate HbF; rare individuals carrying a monoallelic knockout of BCL11A exhibit no known hematologic abnormality and up to 30% circulating HbF. We previously reported de novo knockout of BCL11A using targeted genome editing with engineered zinc finger nucleases (ZFNs) yielding up to 40% HbF in erythroid progeny of edited human CD34 cells in vitro. We now find that the targeted ablation of a single, specific GATAA motif in the BCL11A intronic enhancer does not affect in vitro erythroid differentiation, but reproducibly (n=6) activates fetal globin transcription in erythroid progeny of modified CD34 cells; importantly, at similar levels of on-target marking in CD34+ cells, these effects on fetal globin mRNA are comparable to those resulting from ZFN-driven coding knockout of BCL11A itself. We demonstrate reproducible (n=8), high-efficiency (up to 82%; average, 69%) ZFN-driven marking at the enhancer in peripheral blood mobilized human CD34 cells at clinical production scale (>1e8 cells) in a GMP-compliant setting for which we use a clinical-grade electroporation device to deliver nuclease-encoding transcribed mRNA ex vivo. Using erythroid colony assay genotyping we find that up to 70% of the cells in the resulting population are biallelically modified at the target locus, while ~10% remain wild-type, and find comparably high levels of marking in research-scale preparations of CD34 cells from patients with β-thalassemia. We observe robust long-term (18-24 week) engraftment and multilineage differentiation of genome-edited cells in immunodeficient mice, similar to control cells, and equivalent modification at the targeted enhancer locus  at all timepoints in both differentiated (CD19+, CD3+, CD33+) and more primitive progenitor (CD34+CD38low) cells of human origin purified from bone marrow of long-term-engrafted animals. Our findings support clinical development of enhancer editing as a treatment of the β hemoglobinopathies with autologous hematopoietic stem cell transplant.

 

3234 Clonal Analysis of Human Bone Marrow CD34+ Cells Edited By BCL11A-Targeting Zinc Finger Nucleases Reveals Clinically Relevant Levels of Fetal Globin Expression in Edited Erythroid Progeny

Sickle cell disease (SCD) is one of the most common inherited blood disorders and is caused by a mutation at the adult beta globin gene resulting in substitution of valine for glutamic acid at position 6 in the encoded protein. While SCD can be cured by hematopoietic stem cell transplant (HSCT), complete donor chimerism is not required to achieve clinical benefits. Stable mixed chimerism of 10-15% in bone marrow or peripheral blood nucleated cells with >70% donor-derived RBCs has been reported to achieve transfusion independence and a symptom-free state in a SCD patient. It has also been proposed that SCD can be treated by reactivating developmentally silenced fetal gamma globin to form fetal hemoglobin (alpha2gamma2, HbF), which inhibits polymerization of HbS. The effect of HbF is predicted to be maximal when HbF content per cell exceeds 10 pg (~30% of total Hb). Furthermore, pathology is prevented when protective F cells (>30% HbF per cell) constitute >70% of total RBCs. We hypothesize that in a gene therapy setting, if >15% of SCD patients’ autologous HSCs are programmed to produce protective F cells during erythropoiesis, it will translate into >70% protective F cells in circulation and provide significant alleviation of clinical symptoms. Genome wide association studies have identified BCL11A as a major modifier of HbF levels. Subsequent studies have shown that BCL11A plays a critical role in the fetal to adult globin developmental switch and in repressing fetal globin expression in adult erythroid cells. Conditional inactivation of BCL11A in adult erythroid cells leads to high levels of pan-cellular fetal globin expression and correction of hematologic and pathologic defects in a humanized SCD mouse model. Previously, we have reported that zinc finger nucleases (ZFNs) targeting BCL11A either in the coding region or the GATAA motif in the erythroid-specific enhancer efficiently disrupt the BCL11A locus in human primary CD34⁺ cells following electroporation of ZFN-encoding mRNA. Elevated fetal globin expression in bulk erythroid cultures was observed following disruption. To determine what percentage of HSPCs have been modified and whether the HbF/F cell content has reached the hypothesized therapeutic level, we analyzed erythroid cells clonally derived from ZFN-transfected CD34⁺ cells. Genotype of each clonal culture was determined by deep sequencing and globin production was analyzed by a highly sensitive UPLC method. We found that up to 80% of the BFU-Es had both BCL11A alleles edited, half of which had KO/KO alleles (either out of frame mutations for coding region or elimination of the GATAA motif in the enhancer). BCL11A coding KO/KO cells expressed on average 79.1% ± 12.2% fetal globin (Mean ± SD) whereas GATAA motif enhancer region KO/KO cells expressed approximately 48.4% ± 14.1% fetal globin, in comparison with 14.5% ± 9.6% in WT/WT cells .  These levels of fetal globin should be sufficiently high to confer protection against HbS polymerization in sickle cells. WT/KO cells in both coding and enhancer editing experiments showed an intermediate phenotype with fetal globin averaging 26.9%± 9.9% and 25.79% ± 12.6%, respectively. Interestingly, when background (WT/WT) fetal globin level was subtracted, the fetal globin levels in WT/KO cells are comparable to those observed in patients with BCL11A haploinsufficiency, which average 14.6%± 10.3%. Together, our data demonstrate that genome editing of BCL11A using highly efficient ZFNs can lead to clinically relevant levels of fetal globin expression in KO/KO erythroid cells. If the frequency of KO/KO BFU-Es we observed in vitro reflects the frequency of KO/KO HSCs in bone marrow after autologous transplantation, genome editing of BCL11A has the potential to provide significant clinical benefit for patients with SCD.

4422 In Vivo Genome Editing in Neonatal Mouse Liver Preferentially Utilizes Homology Directed Repair

Genome editing has the potential to provide long-term therapeutic gene expression in vivo. We have previously demonstrated efficient editing in a mouse model of hemophilia B through liver-directed adeno-associated viral vector (AAV) delivery of a zinc finger nuclease (ZFN) pair and a corrective donor. We determined that homology is not necessary to achieve efficient levels of genome editing in adult mice, consistent with the fact that quiescent cells, including adult hepatocytes, are not thought to be amenable to homology directed repair (HDR). As a consequence of the donor containing a splice acceptor, both HDR and homology independent vector integration are capable of driving human factor 9 (hF.IX) expression. In this study we sought to determine whether hF.IX expression in mice treated as neonates, undergoing substantial hepatocyte proliferation, is predominantly the result of HDR or homology independent genome editing. Provided the efficacy is not substantially reduced, an HDR dependent approach would impose additional constraints on targeting.

Treatment of neonatal hF9mut mice (harboring the ZFN target site) with 1x10¹¹ vg AAV8-ZFN and 5x10¹¹ vg AAV8-Donor via retro-orbital injection resulted in a drastic difference in hF.IX expression between donors with and without homology 10 weeks post injection (Homology: 1531 ± 174.5 ng/mL vs. No-homology: 146.1 ± 5.8 ng/mL; n=12 and 7, respectively). We next asked whether HDR could be stimulated even more specifically through the induction of DNA single strand breaks at the target site. We treated neonatal mice with homologous or non-homologous donors, as well as ZFNs or ZFNickases (in which one FokI nuclease domain was inactivated with the D450A mutation). ZFNickases were indeed active, resulting in ~250 ng/mL hF.IX 4 weeks post injection (Figure 1). Interestingly, we could not detect hF.IX in mice treated with ZFNickase and no-homology donor (LOD: 15ng/mL). To rule out the possibility that this was simply due to the lower efficacy of ZFNickases compared to ZFNs, we increased the ZFNickase dose 4 fold. Four weeks post treatment, we observed substantial levels of hF.IX in mice treated with homologous donor (2041 ± 269 ng/mL) and were again unable to detect hF.IX in mice treated with the non-homologous donor (n=10 and 7, respectively). These data point to homology directed repair as the primary mechanism of protein production for genome editing in neonatal mouse liver, and suggest improvements in both efficacy and specificity can be made through deeper understanding of the molecular requirements of this approach.

7. Genome Editing of the Bcl11A Erythroid Specific Enhancer in Bone Marrow Derived Hematopoietic Stem and Progenitor Cells for the Treatment of Sickle Cell Disease

Ablation of Bcl11A could be a viable approach for the treatment of β-hemoglobinopathies such as β-thalassemia and sickle cell disease (SCD), since patients with Bcl11A haploinsufficiency have persistently high levels of fetal hemoglobin (HbF) (up to 30%), which are associated with development of minimal to no disease symptoms.  Genome editing by engineered zinc-finger nucleases that target either the exon 2 (exon ZFN) or the GATA motif of the erythroid specific enhancer (enhancer ZFN) of Bcl11A has been shown to increase HbF level in erythroid progeny from mobilized peripheral hematopoietic stem and progenitor cells (PB-CD34+ HSPCs).  However, peripheral mobilization of CD34+ cells is associated with high risk and currently is not an option for SCD patients.  Therefore, we investigated the efficacy of genome editing of Bcl11A in bone marrow derived CD34+ cells (BM-CD34+ HSPCs). We first established a clinically compatible large-scale process to isolate CD34+ HSPCs from human bone marrow aspirates and to transiently express the ZFN protein by mRNA electroporation.  The CD34+ isolation process resulted in ~ 95% pure CD34+ cells with greater than 90% viability.  Both the exon and the enhancer ZFN drove 50-60% Bcl11A gene editing, resulting in a robust elevation of HbF in the erythroid progeny. Notably, the BM-CD34+ HSPCs were found to contain a small population (10 to 25%) of CD34+CD19+ pro-B cells that were refractory to ZFN transfection under our current electroporation condition.  Since CD34+CD19+ pro-B cells are not expected to contribute to reconstituting the hematopoietic system other than B-cell lineage, the Bcl11A editing efficiency in the multipotent BM-CD34+ HSPC could be even higher.
The engraftment abilities of Bcl11A edited BM-CD34+ cells were then investigated in an immunodeficient NOD/scid/gamma (NSG) mouse model.  At a dose of 1 million cells per mouse, treatment with either the exon ZFN or the enhancer ZFN did not detectably impact engraftment or multi-lineage reconstitution compared with untreated cells.  However, Bcl11A marking in engrafted human cells was found to be markedly higher in the mice treated by the enhancer ZFN than that by the exon ZFN.   The exon ZFN resulted in a strong bias towards in-frame mutations across multi-lineages with the strongest effect observed in the B-cell lineage, suggesting that a threshold level of Bcl11A is required for efficient hematopoietic reconstitution and that cells fully lacking it due to disruption of the coding sequence are at a disadvantage.  In contrast, the enhancer ZFN resulted in comparable Bcl11A marking across all lineages with no apparent selection for cells with a functional GATA sequence.  Collectively, these data indicate that genome editing of the erythroid specific enhancer of Bcl11A in BM-CD34+ promotes HbF reactivation in the erythroid progeny while maintaining the engraftment and multi-lineage repopulating activities of edited BM-CD34+ HSPCs, which supports further clinical development of this approach for the treatment of SCD.

 

 

Bluebird Bio to Present 6 abstracts at ASH

 Six abstracts accepted for presentation –

– Interim data on LentiGlobin in beta-thalassemia show six of six evaluable non-β⁰/β⁰ patients are transfusion-independent as of July 31^st data cut-off; β⁰/β⁰ patients have achieved varying degrees of transfusion reduction as of data cut-off –

    – Median production of corrected HbA^T87Q globin: 5.2 g/dL among patients of all genotypes followed for at least six months in HGB-204 study, as of July 31^st data cut-off –

    – Patient with sickle cell disease from HGB-205 study producing 51.5% anti-sickling hemoglobin at nine months post-treatment –

– Company will discuss ASH abstract data in a conference call today at 8:30 a.m. ET–

http://finance.yahoo.com/news/bluebird-bio-present-lentiglobin-bb305-130000247.html

CRISPR to be Tested in Humans by 2017 says Editas' CEO

The biotechnology startup Editas Medicine intends to begin tests of a powerful new form of gene-repair in humans within two years.
Speaking this week at the EmTech conference in Cambridge, Massachusetts, Editas CEO Katrine Bosley said the company hopes to start a clinical trial in 2017 to treat a rare form of blindness using CRISPR, a groundbreaking gene-editing technology.
If Editas’s plans move forward, the study would likely be the first to use CRISPR to edit the DNA of a person.
CRISPR technology was invented three years ago but is so precise and cheap to use it has quickly spread through biology laboratories. Already, scientists have used it to generate genetically engineered monkeys, and the technique has stirred debate over whether modified humans are next.
Editas is one of several startups, including Intellia Therapeutics and CRISPR Therapeutics, that have plans to use the technique to correct DNA disorders that affect children and adults. Bosley said that because CRISPR can “repair broken genes” it holds promise for treating several thousand inherited disorders caused by gene mistakes, most of which, like Huntington’s disease and cystic fibrosis, have no cure.

Sangamo Mention:
Although the Editas study could be the first for CRISPR in humans, it wouldn’t be the first clinical study of gene editing. An older method called zinc fingers is already in testing to treat HIV infection by the biotechnology company Sangamo Biosciences. But the versatility and ease with which CRISPR can change DNA means it may outpace earlier approaches.


Read more here:
http://www.technologyreview.com/news/543181/crispr-gene-editing-to-be-tested-on-people-by-2017-says-editas/

Highly efficient homology-driven genome editing in human T cells by combining zinc-finger nuclease mRNA and AAV6 donor delivery

1. Jianbin Wang*,
2. Joshua J. DeClercq,
3. Samuel B. Hayward,
4. Patrick Wai-Lun Li,
5. David A. Shivak,
6. Philip D. Gregory,
7. Gary Lee and
8. Michael C. Holmes

- Author Affiliations

1. Sangamo BioSciences, Inc., Richmond, CA 94804, USA

This Article

1. Nucl. Acids Res. (2015) doi: 10.1093/nar/gkv1121 First published online: November 2, 2015

1. This article is Open AccessOA
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Abstract

The adoptive transfer of engineered T cells for the treatment of cancer, autoimmunity, and infectious disease is a rapidly growing field that has shown great promise in recent clinical trials. Nuclease-driven genome editing provides a method in which to precisely target genetic changes to further enhance T cell function in vivo. We describe the development of a highly efficient method to genome edit both primary human CD8 and CD4 T cells by homology-directed repair at a pre-defined site of the genome. Two different homology donor templates were evaluated, representing both minor gene editing events (restriction site insertion) to mimic gene correction, or the more significant insertion of a larger gene cassette. By combining zinc finger nuclease mRNA delivery with AAV6 delivery of a homologous donor we could gene correct 41% of CCR5 or 55% of PPP1R12C (AAVS1) alleles in CD8⁺ T cells and gene targeting of a GFP transgene cassette in >40% of CD8⁺ and CD4⁺ T cells at both the CCR5 and AAVS1 safe harbor locus, potentially providing a robust genome editing tool for T cell-based immunotherapy.