b-globin lentiviral vectors (b-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 b-globin locus control region (LCR) to construct novel ENCODE core sequences. These novel LCR elements were used to design a b-LV of reduced proviral length, termed CoreGA-AS3-FB, produced at higher titers and possessing superior gene transfer to HSPCs when compared to the fulllength parental b-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 b-LV that will be beneficial for gene therapy of SCD by significantly reducing the cost of vector production and extending the vector supply.

Scarring is a dire consequence of acne vulgaris. Particularly, atrophic acne scarring is highly prevalent among young adults, and its physical and psychological effects can persist throughout their lives if left untreated. This literature review will analyze various non-energy-based approaches to treating atrophic acne scarring, emphasizing recent advances within the last 5 to 10 years. To accomplish this, we performed a PubMed search for various acne scar treatments such as chemical peels, dermabrasion, microdermabrasion, subcision, microneedling, punch techniques, dermal fillers, and thread lifting. Our findings and analysis show that there is no panacean solution to treating atrophic acne scars, which explains the evolving trend towards developing unique combinatorial treatments. Although a fair comparison of each treatment approach is difficult to achieve due to the studies’ varying sample sizes, strength of evidence, treatment execution, etc, there still remains a level of consensus on what treatments are well suited for particular scar types.

Summary Lentiviral vectors (LVs) commonly used for the treatment of hemoglobinopathies often have low titers and sub-optimal gene transfer efficiency for human hematopoietic stem and progenitor cells (HSPCs), hindering clinical translation and commercialization for ex vivo gene therapy. We observed that a high percentage of β-globin LV viral genomic RNAs were incomplete toward the 3′ end in packaging cells and in released vector particles. The incomplete vector genomes impeded reverse transcription in target cells, limiting stable gene transfer to HSPCs. By combining three modifications to vector design and production (shortening the vector length to 5.3 kb; expressing HIV-1 Tat protein during packaging; and packaging in PKR−/− cells) there was a 30-fold increase in vector titer and a 3-fold increase in vector infectivity in HSPCs. These approaches may improve the manufacturing of β-globin and other complex LVs for enhanced gene delivery and may facilitate clinical applications.

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 sequenceintrinsic and lineage-specific enhancer function at near-basepair resolution. We demonstrate the power of LV-MPRA in elucidating the boundaries of previously unknown intrinsic enhancer sequences of the human b-globin locus control region. Our approach facilitated the rapid assembly of novel therapeutic b AS3 -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 vectors (LVs) are robust delivery vehicles for gene therapy as they can efficiently integrate transgenes into host cell genomes. However, LVs with lengthy or complex expression cassettes typically are produced at low titers and have reduced gene transfer capacity, creating barriers for clinical and commercial applications. Modifications of the packaging cell line and methods may be able to produce complex vectors at higher titer and infectivity and may improve production of many different LVs. In this study, we identified two host restriction factors in HEK293T packaging cells that impeded LV production, 2 0 -5 0 -oligoadenylate synthetase 1 (OAS1) and low-density lipoprotein receptor (LDLR). Knocking out these two genes separately led to $2-fold increases in viral titer. We created a monoclonal cell line, CRISPRed HEK293T to Disrupt Antiviral Response (CHEDAR), by successively knocking out OAS1, LDLR, and PKR, a previously identified factor impeding LV titers. Packaging in CHEDAR yielded $7-fold increases in physical particles, full-length vector RNA, and vector titers. In addition, overexpressing transcription elongation factors, SPT4 and SPT5, during packaging improved the production of full-length vector RNA, thereby increasing titers by $2fold. Packaging in CHEDAR with over-expression of SPT4 and SPT5 led to $11-fold increases of titers. These approaches improved the production of a variety of LVs, especially vectors with low titers or with internal promoters in the reverse orientation, and may be beneficial for multiple gene therapy applications.