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Introduction: Sleep apnea, a very common condition among post-stroke patients, is associated with worse functional, cognitive, and neurological outcomes after stroke. Little is known about the association between change of sleep apnea and changes in outcome over time. Methods: Associated with the Brain Attack Surveillance in Corpus Christi (BASIC) project, ischemic stroke patients (2016-2019) were offered sleep apnea testing with a portable respiratory monitor (ApneaLink Plus) shortly after stroke and at 3, 6, and 12 months post-stroke. Sleep apnea was quantified using the respiratory event index (REI), the number of apneas plus hypopneas per recording hour. At 3, 6, and 12-months post-stroke, functional outcome was assessed using activities of daily living (ADL)/instrumental activities of daily living (IADL) score (1-4, lower better); cognitive outcome was assessed using the Modified Mini-Mental State Examination (3MS) (0-100, higher better); neurological outcome was assessed using the NIHSS (0-42, lower better). Multivariate mixed models were fitted under a Bayesian approach to obtain random slopes in REI over time and the three outcomes (1-unit change in outcome per month), adjusted for age, sex, ethnicity, pre-stroke function and cognitive status, education, marital status, stroke history, tpa use, recurrent stroke, and depression. Linear regression models assessed the association between random slope of REI and random slopes of the three outcomes. Results: Of the 482 Mexican American or non-Hispanic white participants with at least one REI measurement, in fully adjusted models, improvement in sleep apnea was significantly associated with improvement in NIHSS (0.0094 (95% CI: 0.0070, 0.0118)) but not with changes in ADL/IADL or 3MS. Conclusions: Improvement in sleep apnea paralleled improvement in neurological status over the first year after stroke.

Introduction: Despite their high risk of mortality, recurrent stroke, and dementia, stroke survivors underutilize ACP. In a non-prespecified subgroup analysis of a randomized trial to increase ACP, we compared the efficacy of the PREPARE for Your Care (PREPARE) plus an easy-to-read advance directive (AD) to an AD alone among stroke survivors. Methods: PREPARE is an interactive digital program with easy-to-read, state-specific ADs, and video stories. Stroke survivors in the PREPARE trial were identified using the International Classification of Diseases codes. The primary outcome was new ACP documentation in the electronic medical record after 12 months; secondary outcome was self-reported ACP completion of: (1) signing official papers naming a surrogate decision maker; (2) discussion of patients’ wishes with surrogates; (3) discussion of patients’ wishes with medical providers; and (4) an AD, scored 0-4. We calculated the change from baseline to 12-month follow-up. Mixed-effects regression models were used, adjusted for health literacy (adequate or limited), baseline ACP documentation, and clustering by physician. Results: Of 986 PREPARE trial participants, 91 (9.2%) were stroke survivors and 72 had complete data. The mean age was 64 years (SD, 6.8 years), 47% were women, 74% identified as a minority race/ethnicity, 40% were Spanish-speaking, 49% had limited health literacy, and 41% had ACP before enrollment. There was a trend toward greater ACP documentation in the PREPARE-arm (table). The PREPARE-arm had significantly greater increase in completed ACP elements than the AD-only arm. Discussion: The PREPARE program shows promise to engage ethnically diverse stroke survivors in ACP and should be studied further. Addition of stroke-specific content and engagement of acute stroke survivors in adaptations may increase the impact of the program.

Background: Post-stroke fatigue (PSF) is a common and often disabling symptom, the etiology of which is poorly understood. Sleep-disordered breathing (SDB) is also highly prevalent among stroke survivors. We explored the relationship between SDB and PSF over time in a bi-ethnic cohort. Methods: Ischemic stroke (IS) patients within the Brain Attack Surveillance in Corpus Christi (BASIC) project were identified through active and passive surveillance, and were offered home SDB screening with a well-validated cardiopulmonary sleep apnea testing device, the ApneaLink Plus. The primary exposure was the respiratory event index (REI). The primary outcome was PSF as measured by the SF-36 vitality scale (lower scores reflect greater fatigue). SDB and PSF were measured at baseline, 3, 6, and 12 months post-stroke. A linear regression model, using GEE to account for repeated measures, was used to examine the associations between REI and PSF at each time point adjusting for sociodemographics, stroke severity, depressive symptoms, pre-stroke fatigue, and comorbidities. Results: SDB screening and at least one PSF assessment were completed by 462 IS patients, who were 44% female, 61% Mexican American (MA), 26% non-Hispanic white, 7% Black, and 5% other race/ethnicity. Median NIHSS was 2 (IQR: 1-4). Multivariable regression model results are presented in the Table. Higher REI at 3 months was associated with greater PSF at 3 months (β= -1.87, 95%CI -3.47, -0.27), but there was no association between REI and PSF at 6 or 12 months. Across timepoints, female sex, depressive symptoms at time of assessment, pre-stroke medication for depression, higher levels of pre-stroke fatigue, and vascular risk factor burden were associated with greater PSF, whereas MA ethnicity was associated with less PSF. Conclusions: Higher REI was associated with greater PSF in the early post-stroke period, but no association was observed at 6 months and beyond. MA ethnicity seems to be protective against PSF.

X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency caused by mutations in the CYBB gene, resulting in the inability of phagocytic cells to eliminate infections. To design a lentiviral vector (LV) capable of recapitulating the endogenous regulation and expression of CYBB, a bioinformatics-guided approach was used to elucidate the cognate enhancer elements regulating the native CYBB gene. Using this approach, we analyzed a 600-kilobase topologically associated domain of the CYBB gene and identified endogenous enhancer elements to supplement the CYBB promoter to develop MyeloVec, a physiologically regulated LV for the treatment of X-CGD. When compared with an LV currently in clinical trials for X-CGD, MyeloVec showed improved expression, superior gene transfer to hematopoietic stem and progenitor cells (HSPCs), corrected an X-CGD mouse model leading to complete protection against Burkholderia cepacia infection, and restored healthy donor levels of antimicrobial oxidase activity in neutrophils derived from HSPCs from patients with X-CGD. Our findings validate the bioinformatics-guided design approach and have yielded a novel LV with clinical promise for the treatment of X-CGD.

Hematopoietic stem cell gene therapy is a promising approach for treating disorders of the hematopoietic system. Identifying combinations of cis-regulatory elements that do not impede packaging or transduction efficiency when included in lentiviral vectors has proven challenging. In this study, we deploy LV-MPRA (lentiviral vector-based, massively parallel reporter assay), an approach that simultaneously analyzes thousands of synthetic DNA fragments in parallel to identify sequence-intrinsic and lineage-specific enhancer function at near-base-pair resolution. We demonstrate the power of LV-MPRA in elucidating the boundaries of previously unknown intrinsic enhancer sequences of the human β-globin locus control region. Our approach facilitated the rapid assembly of novel therapeutic βAS3-globin lentiviral vectors harboring strong lineage-specific recombinant control elements capable of correcting a mouse model of sickle cell disease. LV-MPRA can be used to map any genomic locus for enhancer activity and facilitates the rapid development of therapeutic vectors for treating disorders of the hematopoietic system or other specific tissues and cell types.

Lentiviral vector (LV)-based hematopoietic stem cell (HSC) gene therapy is becoming a promising clinical strategy for the treatment of genetic blood diseases. However, the current approach of modifying 1 × 10 8 to 1 × 10 9 CD34 + cells per patient requires large amounts of LV, which is expensive and technically challenging to produce at clinical scale. Modification of bulk CD34 + cells uses LV inefficiently, because the majority of CD34 + cells are short-term progenitors with a limited post-transplant lifespan. Here, we utilized a clinically relevant, immunomagnetic bead (IB)-based method to purify CD34 + CD38 − cells from human bone marrow (BM) and mobilized peripheral blood (mPB). IB purification of CD34 + CD38 − cells enriched severe combined immune deficiency (SCID) repopulating cell (SRC) frequency an additional 12-fold beyond standard CD34 + purification and did not affect gene marking of long-term HSCs. Transplant of purified CD34 + CD38 − cells led to delayed myeloid reconstitution, which could be rescued by the addition of non-transduced CD38 + cells. Importantly, LV modification and transplantation of IB-purified CD34 + CD38 − cells/non-modified CD38 + cells into immune-deficient mice achieved long-term gene-marked engraftment comparable with modification of bulk CD34 + cells, while utilizing ∼7-fold less LV. Thus, we demonstrate a translatable method to improve the clinical and commercial viability of gene therapy for genetic blood cell diseases.

Site-specific correction of a point mutation causing a monogenic disease in autologous hematopoietic stem and progenitor cells (HSPCs) can be used as a treatment of inherited disorders of the blood cells. Sickle cell disease (SCD) is an ideal model to investigate the potential use of gene editing to transvert a single point mutation at the β-globin locus (HBB). We compared the activity of zinc-finger nucleases (ZFNs) and CRISPR/Cas9 for editing, and homologous donor templates delivered as single-stranded oligodeoxynucleotides (ssODNs), adeno-associated virus serotype 6 (AAV6), integrase-deficient lentiviral vectors (IDLVs), and adenovirus 5/35 serotype (Ad5/35) to transvert the base pair responsible for SCD in HBB in primary human CD34+ HSPCs. We found that the ZFNs and Cas9 directed similar frequencies of nuclease activity. Invitro, AAV6 led to the highest frequencies of homology-directed repair (HDR), but levels of base pair transversions were significantly reduced when analyzing cells invivo in immunodeficient mouse xenografts, with similar frequencies achieved with either AAV6 or ssODNs. AAV6 also caused significant impairment of colony-forming progenitors and human cell engraftment. Gene correction in engrafting hematopoietic stem cells may be limited by the capacity of the cells to mediate HDR, suggesting additional manipulations may be needed for high-efficiency gene correction in HSPCs.

Objective: To evaluate a strategy to increase use of the test (Dix-Hallpike test [DHT]) and treatment (canalith repositioning maneuver [CRM]) for Benign Paroxysmal Positional Vertigo (BPPV) in emergency department (ED) dizziness visits. Methods: We conducted a stepped-wedge randomized trial in six EDs. The population was visits with dizziness as a principal reason for the visit. The intervention included educational sessions and decision aid materials. Outcomes were DHT or CRM documentation (primary), head CT use, length-of-stay, admission, and 90-day stroke events. The analysis was multi-level logistic regression with intervention, month, and hospital as fixed effects, and provider as a random effect. We assessed fidelity with monitoring intervention use and semi-structured interviews. Results: We identified 7,635 dizziness visits over 18 months. The DHT or CRM was documented in 1.5% of control visits (45/3077; 95% CI, 1%-1.9%) and 3.5% of intervention visits (159/4558; 95% CI, 3%-4%; difference 2% 95% CI, 1.3%, 2.7%). Head CT use was lower in intervention visits compared with controls (44.0% [1352/3077] vs 36.9% [1682/4558]). No differences were observed in admission or 90-day subsequent stroke risk. In fidelity evaluations, providers who used the materials typically reported positive clinical experiences but provider engagement was low at facilities without an emergency medicine residency program. Conclusions: These findings provide evidence that an implementation strategy of a BPPV-focused approach to ED dizziness visits can be successful and safe in promoting evidence-based care. Absolute rates of DHT and CRM use, however, were still low which relates in part to our broad inclusion criteria for dizziness visits.

β-globin lentiviral vectors (β-LV) have faced challenges in clinical translation for gene therapy of sickle cell disease (SCD) due to low titer and sub-optimal gene transfer to hematopoietic stem and progenitor cells (HSPCs). To overcome the challenge of preserving efficacious expression while increasing vector performance, we used published genomic and epigenomic data available through ENCODE to redefine enhancer element boundaries of the β-globin locus control region (LCR) to construct novel ENCODE core sequences. These novel LCR elements were used to design a β-LV of reduced proviral length, termed CoreGA-AS3-FB, produced at higher titers and possessing superior gene transfer to HSPCs when compared to the full-length parental β-LV at equal MOI. At low vector copy number, vectors containing the ENCODE core sequences were capable of reversing the sickle phenotype in a mouse model of SCD. These studies provide a β-LV that will be beneficial for gene therapy of SCD by significantly reducing the cost of vector production and extending the vector supply.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated system (Cas9)-mediated gene editing of human hematopoietic stem cells (hHSCs) is a promising strategy for the treatment of genetic blood diseases through site-specific correction of identified causal mutations. However, clinical translation is hindered by low ratio of precise gene modification using the corrective donor template (homology-directed repair, HDR) to gene disruption (nonhomologous end joining, NHEJ) in hHSCs. By using a modified version of Cas9 with reduced nuclease activity in G1 phase of cell cycle when HDR cannot occur, and transiently increasing the proportion of cells in HDR-preferred phases (S/G2), we achieved a four-fold improvement in HDR/NHEJ ratio over the control condition in vitro, and a significant improvement after xenotransplantation of edited hHSCs into immunodeficient mice. This strategy for improving gene editing outcomes in hHSCs has important implications for the field of gene therapy, and can be applied to diseases where increased HDR/NHEJ ratio is critical for therapeutic success. Stem Cells 2019;37:284–294

International audience; Sickle cell disease (SCD) is an inherited blood disorder caused by a single amino acid substitution in the beta-globin chain of hemoglobin. Gene therapy is a promising therapeutic alternative, particularly in patients lacking an allogeneic bone marrow (BM) donor. One of the major challenges for an effective gene therapy approach is the design of an efficient vector that combines high-level and long-term beta-globin expression with high infectivity in primary CD34+ cells. Two lentiviral vectors carrying an anti-sickling beta-globin transgene (AS3) were directly compared: the Lenti/betaAS3-FB, and Globe-AS3 with and without the FB insulator. The comparison was performed initially in human BM CD34+ cells derived from SCD patients in an in vitro model of erythroid differentiation. Additionally, the comparison was carried out in two in vivo models: First, an NOD SCID gamma mouse model was used to compare transduction efficiency and beta-globin expression in human BM CD34+ cells after transplant. Second, a sickle mouse model was used to analyze beta-globin expression produced from the vectors tested, as well as hematologic correction of the sickle phenotype. While minor differences were found in the vectors in the in vitro study (2.4-fold higher vector copy number in CD34+ cells when using Globe-AS3), no differences were noted in the overall correction of the SCD phenotype in the in vivo mouse model. This study provides a comprehensive in vitro and in vivo analysis of two globin lentiviral vectors, which is useful for determining the optimal candidate for SCD gene therapy.

Background: Mexican Americans (MAs) have worse stroke outcomes than non-Hispanic whites (NHWs), which could translate into worse quality of life (QOL). Our objective was to investigate ethnic differences in post-stroke QOL across multiple domains in a bi-ethnic community. Methods: Ischemic stroke survivors, identified through the population-based Brain Attack Surveillance in Corpus Christi (BASIC) Project in Nueces County, Texas, participated in in-person interviews 90 days post-stroke from May 2010 - June 2012. The validated short form stroke-specific QOL (SSQOL) was used to assess overall, physical, and psychosocial QOL; scores range from 1-5 with higher scores representing greater QOL. Tobit regression was used to model unadjusted associations between ethnicity and the 3 QOL domains. Models were then adjusted for the following demographic and clinical factors obtained from the medical record and patient interview: age, sex, education, insurance status, marital status, nursing home residence before stroke, prestroke modified Rankin scale, prestroke Informant Questionnaire on Cognitive Decline in the Elderly, initial NIH stroke scale, risk factors, BMI, and a comorbidity index. Results: A total of 335 ischemic strokes with complete data were identified (66% MA, 34% NHW). QOL was lower among MAs compared to NHWs, both overall (mean difference=-0.3, p=0.02) and in the physical domain (mean difference=-0.4, p=0.008); however, there was no difference in the psychosocial domain (p=0.22). After adjustment for demographic and clinical factors, MAs continued to experience poorer QOL compared to NHWs overall (mean difference=-0.5,p=0.049) and in the physical domain (mean difference=-0.3, p=0.006). Conclusions: Ethnic disparities in stroke outcome extend to QOL, with MAs experiencing substantially worse post-stroke QOL than NHWs. Interventions are urgently needed to improve stroke outcomes among the rapidly growing MA stroke population.

Objective: The Stroke Health and Risk Education (SHARE) Project is a cluster-randomized, faith-based, culturally-sensitive, theory-based behavioral intervention trial to reduce key stroke risk factor behaviors. Methods: Ten Catholic churches in Corpus Christi, Texas were randomized to intervention or control group. Subjects were enrolled as friend or family partners who provided reciprocal support to each other. The intervention group received a 1-year multicomponent intervention that included self-help materials (physical activity guide with pedometer, healthy eating guide, blood pressure management photonovella, and motivational short film), 2 tailored newsletters, 5 motivational interviewing counseling calls, and a peer partnership support workshop. Multilevel modeling, accounting for clustering within pairs and parishes, was used to test treatment differences in the average of two measures of change since baseline (ascertained at 6 and 12 months), using standardized questionnaires, in dietary sodium, dietary fruit and vegetable intake, and physical activity of moderate or greater intensity. The primary endpoint was met if any one of the three outcomes was significant based on a pre-specified p-value threshold of 0.05/3. Results: Of 801 subjects who consented, 760 completed baseline data assessments. The median age was 52; 84% were Hispanic, the remainder were non-Hispanic whites; 64% were women. The intervention group had a greater increase in fruit and vegetable intake than the control group (0.25 (95% CI: 0.08, 0.42) cups per day, p = 0.002), a greater decrease in sodium intake (-123.17 (-194.76, -51.59) mg/day, p=0.04), but no difference in change in moderate or greater intensity physical activity (-27.47 (-526.32, 471.38) MET-minutes per week, p=0.56). Conclusion: This cluster-randomized trial was effective in increasing fruit and vegetable intake among Hispanic and non-Hispanic white Catholic parishioners, reaching its primary endpoint. The intervention also seemed to lower sodium intake. Church-based health promotions can be successful in primary stroke prevention efforts.

Obstructive sleep apnea (OSA) is a very common condition in patients with stroke and is found in over half of stroke patients. There is a complex relationship between OSA and stroke, attributable to shared risk factors. There are numerous mechanisms by which OSA may contribute to increased stroke risk, including promotion of atherosclerosis, hypercoagulability, and adverse effects on cerebral hemodynamics. Obstructive sleep apnea is also a risk factor for hypertension, and likely for atrial fibrillation and diabetes, conditions that in turn are risk factors for stroke. OSA is also associated with poor outcomes following stroke. Further epidemiological studies are needed to assess the relationship between OSA and stroke better. Clinical trials using continuous positive airway pressure as a treatment for OSA in stroke patients are needed to determine whether treatment of this condition alters outcome following stroke.

Despite widely varying critical response, 50 years after its original publication C.S. Lewis's series The Chronicles of Narnia remains more popular than ever. One of the reasons for its continuing appeal may be that it has become a functional part of the cultural heritage of its readers and of those it has been read to, replacing the more traditional myths that informed preceding generations. Following a universal pattern — departure from a familiar home, initiation into a larger unknown world, trial and testing, some form of death and renewal, and then finally return and reinvigoration — the Narnia protagonists can be seen as modern incarnations of what Joseph Campbell has labeled the hero with a thousand faces, and as such can serve as models in the process of maturation and development.

Background/Objective: As a primary, pre-specified objective of the population-based Brain Attack Surveillance in Corpus Christi (BASIC) project we studied trends in mortality following ischemic stroke among Mexican Americans (MAs) and non Hispanic whites (NHWs). MAs were previously found to have lower mortality following stroke than NHWs. Methods: We performed active and passive surveillance, and using source documentation, validated all ischemic stroke cases from January, 2000-December, 2010. Deaths were ascertained from the Texas Department of Health through December 31, 2011. Cumulative 30 day and 1 year mortality adjusted for covariates was estimated using log-binomial models with a linear term for year of stroke onset used to model time trends. Pre-specified adjustment factors were: age, sex, prior stroke or TIA, atrial fibrillation, diabetes, heart disease, hypertension, smoking, high cholesterol and stroke severity. Models used data from the entire study period to estimate adjusted mortality among stroke cases in 2000 and 2010, and to calculate projected ethnic differences. Results: There were 1,822 ischemic strokes among NHWs and 2,211 among MAs. Between 2000 and 2010, 30 day and 1 year mortality declined among NHWs, from 8.6% to 5.9% (p=0.14), and 21.7% to 17.0% (p=0.06), respectively. Among MAs, 30 day mortality remained stagnant at 5.6% (p=0.98), and a slight decline from 18.3% to 16.4% was observed for 1 year mortality (p=0.41). While, the ethnic differences in 30 day (p=0.01) and 1 year (p=0.07) mortality were robust in 2000, they were not so in 2010 (30 day, p=0.76; 1 year p=0.78). See figure. MAs are projected to have higher post-stroke mortality by 2022. Conclusions: Overall, mortality following ischemic stroke has declined in the last decade. However, the survival advantage previously documented among MAs vanished by 2010. Further, faster declines in mortality among NHWs than MAs imply a reversal of the survival advantage by approximately 2022.

Background and Purpose— In a previous study, 0.3 and 0.45 mg/kg of intravenous recombinant tissue plasminogen activator (rt-PA) were safe when combined with eptifibatide 75 mcg/kg bolus and a 2-hour infusion (0.75 mcg/kg per minute). The Combined Approach to Lysis Utilizing Eptifibatide and rt-PA in Acute Ischemic Stroke–Enhanced Regimen (CLEAR-ER) trial sought to determine the safety of a higher-dose regimen and to establish evidence for a phase III trial. Methods— CLEAR-ER was a multicenter, double-blind, randomized safety study. Ischemic stroke patients were randomized to 0.6 mg/kg rt-PA plus eptifibatide (135 mcg/kg bolus and a 2-hour infusion at 0.75 mcg/kg per minute) versus standard rt-PA (0.9 mg/kg). The primary safety end point was the incidence of symptomatic intracranial hemorrhage within 36 hours. The primary efficacy outcome measure was the modified Rankin Scale (mRS) score ≤1 or return to baseline mRS at 90 days. Analysis of the safety and efficacy outcomes was done with multiple logistic regression. Results— Of 126 subjects, 101 received combination therapy, and 25 received standard rt-PA. Two (2%) patients in the combination group and 3 (12%) in the standard group had symptomatic intracranial hemorrhage (odds ratio, 0.15; 95% confidence interval, 0.01–1.40; P =0.053). At 90 days, 49.5% of the combination group had mRS ≤1 or return to baseline mRS versus 36.0% in the standard group (odds ratio, 1.74; 95% confidence interval, 0.70–4.31; P =0.23). After adjusting for age, baseline National Institutes of Health Stroke Scale, time to intravenous rt-PA, and baseline mRS, the odds ratio was 1.38 (95% confidence interval, 0.51–3.76; P =0.52). Conclusions— The combined regimen of intravenous rt-PA and eptifibatide studied in this trial was safe and provides evidence that a phase III trial is warranted to determine efficacy of the regimen. Clinical Trial Registration— URL: http://www.clinicaltrials.gov . Unique identifier: NCT00894803.

Coupling Fourier transform infrared spectroscopy with focal plane array detectors at synchrotron radiation sources (SR-FTIR-FPA) has provided a rapid method to simultaneously image numerous biochemical markers in situ at diffraction limited resolution. Since cells and nuclei are well resolved at this spatial resolution, a direct comparison can be made between FTIR functional group images and the histology of the same section. To allow histological analysis of the same section analyzed with infrared imaging, unfixed air-dried tissue sections are typically fixed (after infrared spectroscopic analysis is completed) via immersion fixation. This post fixation process is essential to allow histological staining of the tissue section. Although immersion fixation is a common practice in this filed, the initial rehydration of the dehydrated unfixed tissue can result in distortion of subcellular morphology and confound correlation between infrared images and histology. In this study, vapor fixation, a common choice in other research fields where postfixation of unfixed tissue sections is required, was employed in place of immersion fixation post spectroscopic analysis. This method provided more accurate histology with reduced distortions as the dehydrated tissue section is fixed in vapor rather than during rehydration in an aqueous fixation medium. With this approach, accurate correlation between infrared images and histology of the same section revealed that Purkinje neurons in the cerebellum are rich in cytosolic proteins and not depleted as once thought. In addition, we provide the first direct evidence of intracellular lactate within Purkinje neurons. This highlights the significant potential for future applications of SR-FTIR-FPA imaging to investigate cellular lactate under conditions of altered metabolic demand such as increased brain activity and hypoxia or ischemia.

Lentiviral (LV)-based hematopoietic stem and progenitor cell (HSPC) gene therapy is becoming a promising clinical strategy for the treatment of genetic diseases. Clinical trials are currently underway to treat hemoglobinopathies using a lentivirus expressing anti-sickling β‐ or γ-globin genes. However, clinical scale production of globin vectors has proven difficult due to the large size and complexity of the human β-globin gene expression cassette. Vector products often have low titers and require large manufacturing volumes to treat a single patient. We hypothesized that less vector per patient could be used by further purifying hematopoietic stem cells (HSC) beyond standard CD34+ selection prior to gene modification and transplantation. Here, we have optimized and characterized an immunomagnetic bead (IB) based cell sorting method to enrich CD34+CD38- cells from human bone marrow (BM). Our results suggest we can use clinically available technology (IB-based cell sorting) to achieve a ~10-fold reduction in vector requirements while still retaining the HSC required for clinical benefit after transplant. We first used competitive transplant studies to determine the SCID repopulating activity of different subpopulations of BM CD34+ cells after 2-day ex-vivo culture and LV transduction. The CD34+ BM cells were sorted into 3 intervals of increasing CD38 expression, each marked with a distinct fluorescent LV vector, and competitively transplanted into NSG mice. At >16 weeks post-transplant, >90% of all hCD45+hCD34+ cells in NSG marrow were derived from the lowest ~6% of CD38 negativity. We next optimized small-scale (50-100 mL of BM) enrichment of CD34+CD38- cells using an IB-based cell isolation method. CD38+ cells were first depleted, and CD34+38- cells were subsequently selected. This double-step CD34+CD38- purification non-specifically enriched granulocytes, which represented up to 50% of total isolated cells. Thus, we modified the first step of our protocol to include co-depletion of both CD15+ myeloid cells and CD38+ cells, which increased the purity of CD34+38- cells up to 2-fold. The dual IB-based CD34+CD38- purification and standard CD34+ purification were performed in parallel on 4 independent BM samples. Recovery of CD34+CD38- cells from starting material (MNCs) was assessed by each method. The single-step IB CD34+ purification recovered 91.8±6.5% (mean±SD) of CD34+CD38- cells while double-step IB CD34+CD38- purification recovered 72±1.4% of CD34+CD38- cells. An additional 20±6% of CD34+CD38- cells remained in the CD38+ fraction. When compared to single-step IB CD34+ purification, CD34+CD38- two-step purification reduced the total number of isolated cells by a range of 7.6-17.8-fold. We next compared the long-term engraftment capabilities of IB-purified CD34+ and CD34+CD38- cells transduced with a LV expressing mCitrine. CD34+ or CD34+CD38- cells purified from an equivalent volume of marrow were transplanted into NSG mice at limiting cell doses. Purified cells were transduced at the same cell density and vector concentration, with the CD34+CD38- cell grafts requiring 11.8-fold less LV than CD34+ cells. At 20 weeks, recipients of CD34+ or CD34+CD38- purified cells had similar levels of human chimerism indicating retention of engraftment capacity by the enriched CD34+CD38- cells. Additionally, hCD45+ cells in engrafted NSG mice exhibited similar levels of mCitrine expression (MFI), suggesting that IB enrichment of CD34+CD38- cells does not affect gene transfer into HSC. One potential disadvantage of using purified CD34+CD38- cells in a clinical setting is delayed myeloid recovery in the post-transplant period. NSG mice transplanted with purified CD34+CD38- cells demonstrated reduced circulating human myeloid cells at 3 weeks post-transplant as compared to mice transplanted with CD34+ cells. In order to overcome this potential clinical hurdle, we explored a strategy of adding back non-transduced CD38+ cells, which restored early levels of circulating human myeloid cells. We are currently investigating the effects of adding non-transduced CD38+ cells on long-term engraftment of gene-marked HSC. In summary, we have developed a clinically applicable method to enrich HSCs and reduce vector requirements. This strategy could directly contribute to clinical trials for hemoglobinopathies by addressing the factors currently limiting gene therapy methods. Disclosures No relevant conflicts of interest to declare.

Sickle cell disease (SCD, OMIM 603903) is one the most common inherited blood disorders worldwide, caused by a single amino acid substitution (E6V) in the β-globin chain. SCD is associated with abnormal red cell morphology, hemolytic anemia, vascular occlusion, severe pain, progressive organ damage and reduced life expectancy. Gene therapy by transplantation of autologous, genetically corrected hematopoietic stem cells could be a therapeutic alternative, particularly in patients lacking an allogeneic bone marrow donor. Recent attempts to treat β-thalassemia and SCD with gene therapy showed encouraging results as well as potential limits of a technology based on complex lentiviral vectors for stem cell transduction due to the vector size and complexity, and limited globin synthesis. Designing an efficient vector that combines high-level globin expression with high titer and infectivity remains a formidable challenge. The GLOBE vector carries a β-globin gene under the control of its promoter and a reduced version of the β-globin locus control region (LCR); it was designed to reduce the size and complexity of the LCR while maintaining its enhancer and chromatin opening activity (Miccio et al., PNAS 2008). Based on the GLOBE design, we developed novel LVs for SCD gene therapy, carrying a modified transgene encoding an anti-sickling β-globin gene with 3 amino acid substitutions (AS3), alternative β-globin promoters and a short sequence (FB) with insulating and enhancer-blocking activity in the LTR. The vectors are produced at high titer and reproducibly transduce 60-70% of human BM CD34+ cell-derived clonogenic progenitors at an average VCN of 3. The GLOBE vectors were compared to the Lenti/βAS3-FB vector (Romero, Urbinati et al. JCI 2013), previously developed for a gene therapy clinical trial of SCD. We will present data comparing the different vectors for transduction efficiency in BM CD34+ cells from healthy donors and SCD patients, βAS3-globin transgene expression and anti-sickling activity, in vivo repopulation activity in NSG mice and vector integration profiles in repopulating cells before and after transplantation. This study provides a comprehensive analysis of different LV vectors, useful to determine the optimal candidate for gene therapy of SCD that can provide high transduction of Hematopoietic Stem and Progenitor Cells (HSPCs) as well as maintaining an adequate expression of the anti-sickling globin gene.

Background Obstructive sleep apnea is a risk factor for ischemic stroke and may play a role in post-stroke outcomes. Ascertaining self-reported information about pre-stroke sleep apnea risk in the acute stroke period is challenging as many stroke patients have deficits that hinder communication. We examined agreement between stroke patients and family members (proxy) with respect to pre-stroke risk of sleep apnea and hypothesized that agreement would be good. Methods Patient-proxy pairs (n=38) were interviewed as part of the Brain Attack Surveillance in Corpus Christi Project from May 2010 - April 2011. The Berlin Questionnaire was used to measure high risk of sleep apnea defined as at least two of the following: 1) snoring behaviors and witnessed apneas 2) daytime sleepiness and 3) hypertension or obesity. Patient-proxy agreement was assessed using a kappa coefficient. Results Average patient age was 70 (SD=12 years), 68% (n=26) were Mexican American and 32% (n=12) non-Hispanic white. An equal number of male and female patients participated (n=19), and average NIH Stroke Scale value at admission was 5.5 (SD = 6.4). Proxies were spouses (n=24) or children (n=14). Proxies had known patients for an average of 40 years (SD=15) and spoke with the patient daily (n=37) or weekly (n=1). Forty percent of patients (n=15) self-identified as high risk for sleep apnea and 52% of proxies (n=20) identified patients as high risk. Patient-proxy agreement for high risk of sleep apnea was fair (kappa=0.43; table 1 ) and was the same for both spouses and children. Conclusions Proxies can be used to assess pre-stroke risk of sleep apnea when a stroke patient is unable to participate as agreement for high risk of sleep apnea was fair. Proxies reported a high risk for sleep apnea with greater frequency than patients, suggesting there may be differences in observation of specific high-risk characteristics between proxies and patients. Table 1. Patient-Proxy Agreement for Risk of Sleep Apnea (n=38) Patient Risk Assessment Proxy Risk Assessment Total Low High Low 15 8 23 High 3 12 15 Total 18 20 38

Background: objective was set to describe and examine physiological and self-reported indices of tolerance of a standing tilt table protocol (STTP) among patients following an acute stroke. Design: Prospective, observational pilot study of patients admitted to a stroke unit of a single academic medical center. Methods: A clinical protocol for the use of the tilt table was developed and applied to subjects in the acute phase following a stroke. The protocol involved a step-wise process to gradually raise the subject into a standing position with lower extremities in a weight bearing position on the tilt table platform, at 10 degree intervals from 60 to 90 degrees. The STTP clinical indices of tolerance were operationally defined as: Systolic blood pressure (SBP) maintained within 20 mmHg of baseline, diastolic blood pressure (DBP) maintained within 10 mmHg of baseline, heart rate (HR) maintained within 10 beats per minute of baseline, oxygen saturation (SpO 2 ) maintained at 90% or above while on supplemental O 2 or room air, absence of pallor. Subjective indices of tolerance were operationally defined as: rating of perceived exertion (RPE) < 15 per Borg Rating of Perceived Exertion Scale, pain < 5 using a numerical pain rating scale ranging from 0 to 10, and absence of angina, dizziness, nausea, dyspnea, or request to discontinue the STTP. No single measure was considered to be of primary importance, and any value falling outside the predetermined parameter was considered indicative of intolerance. Overall, tolerance of the STTP was defined as the ability to sustain 60 degrees or greater of tilt table inclination (minimum of 87% total weight bearing) for a minimum 5 minutes, without signs or symptoms of intolerance. Specifically, frequencies of the highest angle achieved, the duration of standing time tolerated, and physiologic response were recorded. Results: Thirty-six patients with ischemic or hemorrhagic stroke (22 females, 14 males) aged 24 to 87 years (62 + 16 years) enrolled. Fifty three percent of subjects (N = 19) attained 60 degrees or higher on the standing tilt table. Ten subjects were unable to achieve 45 degrees. Subjects tolerated a mean total standing time at or above 60 degrees of 8.9 + 7.2 minutes. All subjects met tolerance criteria for dyspnea, pain, pallor, perceived effort, and nausea at 60 degrees or higher. All subjects were tolerant by heart rate criteria at 70, 80 and 90 degrees; 96% were tolerant at 60 degrees. For diastolic blood pressure, tolerance ranged from 72% (80 degrees) to 90% (70 degrees). For systolic blood pressure, tolerance ranged from 84% (70 degrees) to 100% (90 degrees). Oxygen saturation tolerance was achieved in 100% of subjects at each angle. Conclusion: This pilot study suggests that use of a tilt table is well tolerated among patients in the acute stroke phase, and may be an effective tool for introducing early mobilization to a medically fragile patient population.