Reactivating Fetal Hemoglobin Expression in Human Adult Erythroblasts Through BCL11A Knockdown Using Targeted Endonucleases

http://www.nature.com/mtna/journal/v5/n8/full/mtna201652a.html

Carmen F Bjurström¹, Michelle Mojadidi¹, John Phillips², Caroline Kuo³, Stephen Lai¹, Georgia R Lill¹, Aaron Cooper¹, Michael Kaufman¹, Fabrizia Urbinati¹, Xiaoyan Wang⁴, Roger P Hollis¹ and Donald B Kohn^1,3

1. ¹Department of Microbiology, Immunology, & Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA
2. ²Howard Hughes Medical Institute, University of California, Los Angeles, Los Angeles, California, USA
3. ³Department of Pediatrics, University of California, Los Angeles, Los Angeles, California, USA
4. ⁴Department of General Internal Medicine and Health Services Research, University of California, Los Angeles, Los Angeles, California, USA

Correspondence: Donald B Kohn, Department of Microbiology, Immunology, & Molecular Genetics, University of California, Los Angeles, 3163 Terasaki Life Sciences Building (TLSB) 610 Charles E. Young Drive, East, Los Angeles, California 90095–7364, USA. E-mail: dkohn@mednet.ucla.edu

Received 17 April 2016; Accepted 18 April 2016

Abstract

We examined the efficiency, specificity, and mutational signatures of zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 systems designed to target the gene encoding the transcriptional repressor BCL11A, in human K562 cells and human CD34⁺ progenitor cells. ZFNs and TALENs were delivered as in vitro transcribed mRNA through electroporation; CRISPR/Cas9 was codelivered by Cas9 mRNA with plasmid-encoded guideRNA (gRNA) (pU6.g1) or in vitro transcribed gRNA (gR.1). Analyses of efficacy revealed that for these specific reagents and the delivery methods used, the ZFNs gave rise to more allelic disruption in the targeted locus compared to the TALENs and CRISPR/Cas9, which was associated with increased levels of fetal hemoglobin in erythroid cells produced in vitro from nuclease-treated CD34⁺ cells. Genome-wide analysis to evaluate the specificity of the nucleases revealed high specificity of this specific ZFN to the target site, while specific TALENs and CRISPRs evaluated showed off-target cleavage activity. ZFN gene-edited CD34⁺ cells had the capacity to engraft in NOD-Prkdc^SCID-IL2Rγ^null mice, while retaining multi-lineage potential, in contrast to TALEN gene-edited CD34⁺ cells. CRISPR engraftment levels mirrored the increased relative plasmid-mediated toxicity of pU6.g1/Cas9 in hematopoietic stem/progenitor cells (HSPCs), highlighting the value for the further improvements of CRISPR/Cas9 delivery in primary human HSPCs.

FierceBiotech Reports: Shire Abandons Baxalta Aquired Hemophilia Program

From the FierceBiotech website:
http://www.fiercebiotech.com/biotech/shire-cans-baxalta-hemophilia-b-gene-therapy-thinning-field-for-uniqure-spark

Shire ($SHPG) has scrapped plans to further develop BAX 335, a hemophilia B gene therapy it gained in its $32 billion takeover of Baxalta. The move will see Shire focus its attention on a preclinical gene therapy program it thinks has a better chance of success, clearing the path for uniQure ($QURE) and Spark Therapeutics ($ONCE) to beat their bigger rival to market.
Baxalta had advanced the gene therapy as far as a Phase I/II clinical trial, early data from which were released more than one year ago. Those results showed the gene therapy, which uses an AAV8 vector to deliver factor IX (FIX) Padua, elevated the FIX activity of one participant by 20% to 25% for a full year. Yet, while this represented a success for this one patient over that period of time, the overall, longer-term dataset has failed to convince Shire that BAX 335 is worth pursuing.
“The expression was good but it was a little inconsistent between different patients. And, with time for some patients, the level of expression decreased,” Philip Vickers, head of R&D at Shire, said on a conference call with investors to discuss the company’s second-quarter results.
Shire plans to use the experience gained in the clinic to shape a preclinical program. Vickers said the team has an idea of some of the technical factors that could account for the inconsistency and slide in activity over time. And, with the Phase I/II trial showing that when the AAV8 vector works, it works well, Shire thinks it has the makings of an effective asset. Baxalta, through its then parent company Baxter ($BAX), acquired the AAV8 vector in the $70 million takeover of Chatham Therapeutics.
The decision to scrap BAX 335 removes one of the more advanced riders from the congested race to bring a hemophilia B gene therapy to market. As recently as last month, Baxalta was revising the list of clinical trial sites in the Phase I/II study and BAX 335 was still penciled in to start Phase III this year. Now, Shire has pulled the plug on the trial, shortening the odds that one of the biotechs leading the pack will bring a hemophilia B gene therapy to market before their bigger rival.
UniQure and Spark have both delivered some clinical data on their gene therapies, while Dimension Therapeutics ($DMTX) and Sangamo Biosciences ($SGMO) are closing in on that point, too. Of the group, uniQure is seen by investors as having the most to gain from the scrapping of BAX 335. The share price of the Dutch biotech, which has taken repeated hits over the past year, rose 8.5% on the day Shire revealed its decision. Dimension, Sangamo and Spark all closed down.