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Hemophilia B is an X-linked recessive bleeding disorder that affects approximately 1:20,000 males. It is caused by mutations in the F9gene that encodes for the factor IX (FIX) protein, an essential enzyme in the coagulation cascade. The current recommended therapy for hemophilia B patients is prophylactic administration of exogenous FIX derived from recombinant protein. The short biological half-life of FIX requires frequent infusions to maintain therapeutic level. More recently, an adeno-associated virus (AAV) vector-based gene therapy, etranacogene dezaparvovec, has been approved for some adults as a potential new option. Despite these advances, there remains a significant unmet medical need in hemophilia B. Intravenous infusions of weekly (or more frequently, 3 times per week) factor replacements are an enormous burden to patients and their caregivers. Gene therapy, while a promising option for some, carries potential for significant risk. Importantly, the limitations associated with increasing the dose of FIX needed to treat pediatric patients as they age make immunogenic AAV-based gene therapy inappropriate for this patient population. Terminally differentiated human plasma cells derived from genetically engineered B cells (termed B Cell Medicines, BeCMs), potentially offer natural longevity (persisting for decades), capacity for high levels of protein secretion (thousands of Ig molecules/cell/sec), the ability to engraft without host preconditioning, and the ability to re-dose, making them an attractive platform for the sustained supply of biologics where continuous dosing is required to achieve therapeutic benefit. We have developed an ex vivoprecision engineered BeCM platform with modularity and broad therapeutic utility. In this study, primary human B cells were isolated, activated, and engineered by CRISPR/Cas9 genome editing followed by AAV-mediated homology directed repair (HDR) insertion of human F9gene into the C-C chemokine receptor type 5 (CCR5) safe harbor locus. The cells were then further expanded and differentiated towards the plasma cell lineage, resulting in FIX-producing BeCMs. We achieved approximately 40% targeted integration as measured by droplet digital PCR (ddPCR). Engineered BeCMs secreted up to 60 ng/1e6 cells/hour of FIX protein, approaching 40% of IgG secretion rate as measured by ELISA. BeCM-produced FIX was analyzed by LC-MS which demonstrated gamma carobxylation of FIX protein Gal-domain. Vitamin K-dependent activated partial thromboplastin time (aPTT), using the one stage clotting assay, was employed to verify biological activity of BeCM-produced FIX. Similarly, FIX-expressing BeCMs exhibited vitamin K-dependent activity in vitroin the chromogenic assay. FIX-expressing BeCMs were transferred into immunodeficient NOG-hIL6 mice, with FIX production demonstrated at least 20 weeks in vivo. The safety of FIX-expressing BeCMs has been characterized based on 28-day and 5-month in vivostudies in NOG-hIL6 mice. Neither abnormal clinical observations nor mortality were observed in those studies. Biodistribution of the FIX expressing BeCM product was assessed using a qPCR assay. The qPCR data confirms the expected biodistribution of FIX-expressing BeCMs, which engraft and persist in bone marrow tissue over time. In summary, we have developed an ex vivoprecision gene engineered B cell medicine that produces active and sustained levels of FIX for the treatment of hemophilia B. The potential therapeutic application of this unique biologic delivery system could afford a novel treatment modality for hemophilia B.

Here we show that acute myeloid leukemia (AML) cells require the BRD9 subunit of the SWI−SNF chromatin-remodeling complex to sustain MYC transcription, rapid cell proliferation and a block in differentiation. Based on these observations, we derived small-molecule inhibitors of the BRD9 bromodomain that selectively suppress the proliferation of mouse and human AML cell lines. To establish these effects as on-target, we engineered a bromodomain-swap allele of BRD9 that retains functionality despite a radically altered bromodomain pocket. Expression of this allele in AML cells confers resistance to the antiproliferative effects of our compound series, thus establishing BRD9 as the relevant cellular target. Furthermore, we used an analogous domain-swap strategy to generate an inhibitor-resistant allele of EZH2. To our knowledge, our study provides the first evidence for a role of BRD9 in cancer and reveals a simple genetic strategy for constructing resistance alleles to demonstrate on-target activity of chemical probes in cells.

The bromodomain and extraterminal (BET) protein BRD4 can physically interact with the Mediator complex, but the relevance of this association to the therapeutic effects of BET inhibitors in cancer is unclear. Here, we show that BET inhibition causes a rapid release of Mediator from a subset of cis-regulatory elements in the genome of acute myeloid leukemia (AML) cells. These sites of Mediator eviction were highly correlated with transcriptional suppression of neighboring genes, which are enriched for targets of the transcription factor MYB and for functions related to leukemogenesis. A shRNA screen of Mediator in AML cells identified the MED12, MED13, MED23, and MED24 subunits as performing a similar regulatory function to BRD4 in this context, including a shared role in sustaining a block in myeloid maturation. These findings suggest that the interaction between BRD4 and Mediator has functional importance for gene-specific transcriptional activation and for AML maintenance.

MUSIC MAKING (PLAYING AN INSTRUMENT OR SINGING) is a multimodal activity that involves the integration of auditory and sensorimotor processes. The ability to sing in humans is evident from infancy, and does not depend on formal vocal training but can be enhanced by training. Given the behavioral similarities between singing and speaking, as well as the shared and distinct neural correlates of both, researchers have begun to examine whether singing can be used to treat some of the speech-motor abnormalities associated with various neurological conditions. This paper reviews recent evidence on the therapeutic effects of singing, and how it can potentially ameliorate some of the speech deficits associated with conditions such as stuttering, Parkinson's disease, acquired brain lesions, and autism. By reviewing the status quo, it is hoped that future research can help to disentangle the relative contribution of factors to why singing works. This may ultimately lead to the development of specialized or "gold-standard" treatments for these disorders, and to an improvement in the quality of life for patients.