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X-linked lymphoproliferative disease (XLP1) results from SH2D1A gene mutations affecting the SLAM-associated protein (SAP). A regulated lentiviral vector (LV), XLP-SMART LV, designed to express SAP at therapeutic levels in T, NK, and NKT cells, is crucial for effective gene therapy. We experimentally identified 34 genomic regulatory elements of the SH2D1A gene and designed XLP-SMART LVs to emulate the lineage and stage-specific control of SAP. We screened them for their on-target enhancer activity in T, NK, and NKT cells and their off-target enhancer activity in B cell and myeloid populations. In combination, three enhancer elements increased SAP promoter expression up to 4-fold in on-target populations in vitro. NSG-Tg(Hu-IL15) xenograft studies with XLP-SMART LVs demonstrated up to 7-fold greater expression in on-target cells over a control EFS-LV, with no off-target expression. The XLP-SMART LVs exhibited stage-specific T and NK cell expression in peripheral blood, bone marrow, spleen, and thymic tissues (mimicking expression patterns of SAP). Transduction of XLP1 patient CD8+ T cells or BM CD34+ cells with XLP-SMART LVs restored restimulation-induced cell death and NK cytotoxicity to wild-type levels, respectively. These data demonstrate that it is feasible to create a lineage and stage-specific LV to restore the XLP1 phenotype by gene therapy.

A major limitation of gene therapy for sickle cell disease (SCD) is the availability and access to a potentially curative one-time treatment, due to high treatment costs. We have developed a high-titer bifunctional lentiviral vector (LVV) in a vector backbone that has reduced size, high vector yields, and efficient gene transfer to human CD34+ hematopoietic stem and progenitor cells (HSPCs). This LVV contains locus control region cores expressing an anti-sickling βAS3-globin gene and two microRNA-adapted short hairpin RNA simultaneously targeting BCL11A and ZNF410 transcripts to maximally induce fetal hemoglobin (HbF) expression. This LVV induces high levels of anti-sickling hemoglobins (HbAAS3 + HbF), while concurrently decreasing sickle hemoglobin (HbS). The decrease in HbS and increased anti-sickling hemoglobin impedes deoxygenated HbS polymerization and red blood cell sickling at low vector copy per cell in transduced SCD patient CD34+ cells differentiated into erythrocytes. The dual alterations in red cell hemoglobins ameliorated the SCD phenotype in the SCD Berkeley mouse model in vivo. With high titer and enhanced transduction of HSPC at a low multiplicity of infection, this LVV will increase the number of patient doses of vector from production lots to decrease costs and help improve accessibility to gene therapy for SCD.

ContextAdvance care planning (ACP) is underutilized, especially among Black Americans. Yet, no ACP interventions have been tested at the community level.ObjectivesWithin an established academic and community partnership, we sought to determine whether ACP is a community-identified need and if so, to conduct a pilot study of an evidence-based ACP program, PREPARE (PrepareForYourCare.org).MethodsWe conducted open discussions and in-depth interviews to determine the relevance of ACP to the community. We then conducted a pre- to 3-week postpilot study of a virtual peer facilitated brief session to introduce ACP and encourage participants to engage with PREPARE. We conducted thematic content analysis for qualitative data and used paired t-tests to assess within-participant changes in the validated ACP Engagement Survey measured on a 1-5 scale (5 = greatest engagement).ResultWe conducted two discussion groups with community leaders (n = 12) and key informant interviews (n = 6), including leaders in aging, public health, health care and faith. We concluded that ACP is a community priority. In the pilot study, we enrolled 13 Black Americans; 85% were women and the mean age was 59.7 years (SD 15.1). There was a trend toward increased ACP engagement after the peer facilitated PREPARE (mean 3.2 (SD 0.6) pre vs. 3.5 (SD 0.6) post, paired t-test P = 0.06). All participants found the intervention to be acceptable and were satisfied with it.ConclusionCommunity members identified ACP as important for their community. Peer facilitated PREPARE program is a promising community-based strategy to increase engagement in ACP and may promote health equity.

Objective: We examined clinical trial knowledge and attitudes, and their relationship with willingness to participate in COVID-19 vaccine trials, and willingness to accept a COVID-19 vaccine among college students. Participants: 331 undergraduates: mean age 25; 72% women; and 78% white. Methods: We administered an online, anonymous survey to undergraduate students in July, 2020, during the COVID-19 pandemic. Results: The mean clinical trial knowledge score was 65% (SD = 16) correct. The mean attitudes toward clinical trials score (1 most negative: 5 most positive) was 3.3 (SD = 0.5). Attitudes toward clinical trials were associated with likelihood of COVID-19 trial participation (positive 76% vs. negative 35%, p = 0.001) and a trend toward likelihood of accepting a COVID-19 vaccine if available (positive 89% vs. negative 67%, p = 0.066). Conclusions: General clinical trial knowledge and attitudes appear to be important targets for educational interventions. Furthermore, fostering positive attitudes may lead to improved COVID-19 trial participation and vaccine uptake.

The coronavirus 2019 (COVID-19) pandemic requires drastic changes in allocation of resources, which can affect the delivery of stroke care, and many providers are seeking guidance. As caregivers, we are guided by 3 distinct principles that will occasionally conflict during the pandemic: (1) we must ensure the best care for those stricken with COVID-19, (2) we must provide excellent care and advocacy for patients with cerebrovascular disease and their families, and (3) we must advocate for the safety of health care personnel managing patients with stroke, with particular attention to those most vulnerable, including trainees. This descriptive review by a diverse group of experts in stroke care aims to provide advice by specifically addressing the potential impact of this pandemic on (1) the quality of the stroke care delivered, (2) ethical considerations in stroke care, (3) safety and logistic issues for providers of patients with stroke, and (4) stroke research. Our recommendations on these issues represent our best opinions given the available information, but are subject to revision as the situation related to the COVID-19 pandemic continues to evolve. We expect that ongoing emergent research will offer additional insights that will provide evidence that could prompt the modification or removal of some of these recommendations.

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.

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.

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. In vitro, AAV6 led to the highest frequencies of homology-directed repair (HDR), but levels of base pair transversions were significantly reduced when analyzing cells in vivo 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.

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.

Poly acrylic acid (PAA) is a polymer and a derivative of acrylic acid that is a superabsorbent, being able to absorb and retain water, and swell many times beyond its original volume. This property classifies it into a group of polymers called hydrogels. Hydrogels are being investigated in emerging applications such as drug delivery, biosensors, tissue engineering, wound healing bandages, and more. The ability to lithographically pattern hydrogel materials to specific dimensions at the micro and nanoscales can be very useful in devices and sensors. Limited work has been done on characterizing PAA for lithographic purposes, and so, this work investigates the ability to pattern PAA by electron beam lithography (EBL). PAA is interesting in that its carrier solvent, developer, and remover are all water alone, which may make it attractive for processes or materials that cannot tolerate solvents, acids, and bases used with other common EBL resists. PAA behaves as a negative tone resist with a relatively low base dose of 75 μC/cm2 at 100 kV acceleration voltage. The resolution is limited to 1.8 μm due to a low contrast of 1.29. However, PAA may still have many uses at that resolution where the positioning and dimension control of a hydrogel could be useful. Furthermore, PAA can successfully be used for pattern transfer with either a metal liftoff process or a silicon plasma etch. [ABSTRACT FROM AUTHOR]

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 × 108 to 1 × 109 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.

We previously found that chronic adenosine A1 receptor stimulation with N6-Cyclopentyladenosine increased α-synuclein misfolding and neurodegeneration in a novel α-synucleinopathy model, a hallmark of Parkinson's disease. Here, we aimed to synthesize a dimer caffeine-indan linked by a 6-carbon chain to cross the blood–brain barrier and tested its ability to bind α-synuclein, reducing misfolding, behavioral abnormalities, and neurodegeneration in our rodent model. Behavioral tests and histological stains assessed neuroprotective effects of the dimer compound. A rapid synthesis of the 18F-labeled analogue enabled Positron Emission Tomography and Computed Tomography imaging for biodistribution measurement. Molecular docking analysis showed that the dimer binds to α-synuclein N- and C-termini and the non-amyloid-β-component (NAC) domain, similar to 1-aminoindan, and this binding promotes a neuroprotective α-synuclein "loop" conformation. The dimer also binds to the orthosteric binding site for adenosine within the adenosine A1 receptor. Immunohistochemistry and confocal imaging showed the dimer abolished α-synuclein upregulation and aggregation in the substantia nigra and hippocampus, and the dimer mitigated cognitive deficits, anxiety, despair, and motor abnormalities. The 18F-labeled dimer remained stable post-injection and distributed in various organs, notably in the brain, suggesting its potential as a Positron Emission Tomography tracer for α-synuclein and adenosine A1 receptor in Parkinson's disease therapy. [ABSTRACT FROM AUTHOR]