Multiplex gene editing to promote cell survival using low-pH clustered regularly interspaced short palindromic repeats activation (CRISPRa) gene perturbation.

BACKGROUND AIMS: Lower back pain is the leading cause of disability worldwide and is often linked to degenerative disc disease (DDD), the breakdown of intervertebral discs. The majority of treatment options for DDD are palliative, with clinicians prescribing medication or physical therapy to return the patient to work. Cell therapies are promising treatment options with the potential to restore functional physiological tissue and treat the underlying causes of DDD. DDD is characterized by biochemical changes in the microenvironment of the disc, including changes in nutrient levels, hypoxia, and changes in pH. Stem cell therapies are promising therapies to treat DDD, but the acidic environment in a degenerating disc significantly hinders the viability of stem cells, affecting their efficacy. Clustered regularly interspaced short palindromic repeats (CRISPR) systems allow us to engineer cell phenotypes in a well-regulated and controlled manner. Recently, CRISPR gene perturbation screens have assessed fitness, growth and provided a means for specific cell phenotype characterization. METHODS: In this study, we use a CRISPR-activation (a) gene perturbation screen to identify gene upregulation targets that enhance adipose-derived stem cell survival in acidic culture conditions. RESULTS: We identified 1213 prospective pro-survival genes and systematically narrowed these down to 20 genes for validation. We further narrowed down our selection to the top five prospective genes using Cell Counting Kit-8 cell viability assays in naïve adipose-derived stem cells and ACAN/Col2 CRISPRa upregulated stem cells. Finally, we examined the extracellular matrix-producing abilities of multiplex ACAN/Col2-pro-survival edited cells in pellet culture. CONCLUSIONS: Using the results from the CRISPRa screen, we are able to engineer desirable cell phenotypes to improve cell viability for the potential treatment of DDD and other disease states that expose cell therapies to acidic environments, while also providing broader knowledge on genes regulating low-pH cell survival.

Near-infrared light-responsive hydrogels for on-demand dual delivery of proangiogenic growth factors.

Achieving precise spatiotemporal control over the release of proangiogenic factors is crucial for vasculogenesis, the process of de novo blood vessel formation. Although various strategies have been explored, there is still a need to develop cell-laden biomaterials with finely controlled release of proangiogenic factors at specific locations and time points. We report on the developed of a near-infrared (NIR) light-responsive collagen hydrogel comprised of gold nanorods (GNRs)-conjugated liposomes containing proangiogenic growth factors (GFs). We demonstrated that this system enables on-demand dual delivery of GFs at specific sites and over selected time intervals. Liposomes were strategically formulated to encapsulate either platelet-derived growth factor (PDGF) or vascular endothelial growth factor (VEGF), each conjugated to gold nanorods (GNRs) with distinct geometries and surface plasmon resonances at 710 nm (GNR710) and 1064 nm (GNR1064), respectively. Using near infrared (NIR) irradiation and two-photon (2P) luminescence imaging, we successfully demonstrated the independent release of PDGF from GNR710 conjugated liposomes and VEGF from GNR1064-conjugated liposomes. Our imaging data revealed rapid release kinetics, with localized PDGF released in approximately 4 min and VEGF in just 1 and a half minutes following NIR laser irradiation. Importantly, we demonstrated that the release of each GF could be independently triggered using NIR irradiation with the other GF formulation remaining retained within the liposomes. This light-responsive collagen hydrogels holds promise for various applications in regenerative medicine where the establishment of a guided vascular network is essential for the survival and integration of engineered tissues. STATEMENT OF SIGNIFICANCE: In this study, we have developed a light-responsive system with gold nanorods (GNRs)-conjugated liposomes in a collagen hydrogel, enabling precise dual delivery of proangiogenic growth factors (GFs) at specific locations and timepoints. Liposomes, containing platelet-derived growth factor (PDGF) or vascular endothelial growth factor (VEGF), release independently under near- infrared irradiation. This approach allows external activation of desired GF release, ensuring high cell viability. Each GF can be triggered independently, retaining the other within the liposomes. Beyond its application in establishing functional vascular networks, this dual delivery system holds promise as a universal platform for delivering various combinations of two or more GFs.

BRCA1 and BRCA2 Mutation Testing in Young Women With Breast Cancer.

IMPORTANCE: BRCA testing is recommended for young women diagnosed as having breast cancer, but little is known about decisions surrounding testing and how results may influence treatment decisions in young patients. OBJECTIVES: To describe the use of BRCA testing and to evaluate how concerns about genetic risk and use of genetic information affect subsequent treatment decisions in young women with breast cancer. DESIGN, SETTING, AND PARTICIPANTS: Cross-sectional analysis of data collected following the opening of the study to accrual from October 10, 2006, through December 31, 2014, as part of the Helping Ourselves, Helping Others: Young Women's Breast Cancer Study, an ongoing prospective cohort study. Study participants included 897 women aged 40 years and younger at breast cancer diagnosis from 11 academic and community medical centers. MAIN OUTCOMES AND MEASURES: Frequency and trends in the use of BRCA testing and how genetic information is used to make treatment decisions among women who test positive vs negative for a BRCA mutation. RESULTS: A total of 780 (87.0%) of 897 women reported BRCA testing by 1 year after breast cancer diagnosis (mean age at diagnosis, 35.3 vs 36.9 years for untested women; P < .001), with the frequency of testing increasing among women diagnosed from August 1, 2006, through December 31, 2013. Of 39 women who were diagnosed as having breast cancer in 2006, 30 (76.9%) reported testing. In 2007, a slightly lower percentage of women (87 of 124 [70.2%]) reported testing; however, the proportion tested increased each subsequent year, with 141 (96.6%) of 146 and 123 (95.3%) of 129 women diagnosed as having breast cancer in 2012 and 2013, respectively, reporting BRCA testing (P < .001). Among untested women, 37 (31.6%) of 117 did not report discussion of the possibility that they might have a mutation with a physician and/or genetic counselor, and 43 (36.8%) of 117 were thinking of testing in the future. A total of 248 (29.8%) of 831 women said that knowledge or concern about genetic risk influenced treatment decisions; among these women, 76 (86.4%) of 88 mutation carriers and 82 (51.2%) of 160 noncarriers chose bilateral mastectomy (P < .001). Fewer women reported that systemic treatment decisions were influenced by genetic risk concern. CONCLUSIONS AND RELEVANCE: Rates of BRCA1 and BRCA2 mutation testing are increasing in young women with breast cancer. Given that knowledge and concern about genetic risk influence surgical decisions and may affect systemic therapy trial eligibility, all young women with breast cancer should be counseled and offered genetic testing, consistent with the National Comprehensive Cancer Network guidelines.