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.

The recent emergence of engineered cellular therapies, such as Chimeric antigen receptor (CAR) CAR T and T cell receptor (TCR) engineered T cells, has shown great promise in the treatment of various cancers. These agents aggregate and expand exponentially at the tumor site, resulting in potent immune activation and tumor clearance. Moreover, the ability to elaborate these cells with therapeutic agents, such as antibodies, enzymes, and immunostimulatory molecules, presents an unprecedented opportunity to specifically modulate the tumor microenvironment through cell-mediated drug delivery. This unique pharmacology, combined with significant advances in synthetic biology and cell engineering, has established a new paradigm for cells as vectors for drug delivery. Targeted cellular micropharmacies (TCMs) are a revolutionary new class of living drugs, which we envision will play an important role in cancer medicine and beyond. Here, we review important advances and considerations underway in developing this promising advancement in biological therapeutics.

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.

A major problem with mRNA therapeutics is that the mRNA is usually degraded within a few hours after entering the cytosol. This limits the duration and total amount of protein expression. New approaches for in vitro synthesis of circular mRNA have allowed increased levels and duration of protein synthesis from mRNA therapeutics due to the long half-life of circular mRNA. However, it remains difficult to genetically encode circular mRNAs in mammalian cells, which makes it difficult to use circular mRNA in cell-derived therapeutics. Here we describe the adaptation of the Tornado (Twister-optimized RNA for durable overexpression) system to achieve in-cell synthesis of circular mRNAs. We screen different promoters and internal ribosomal entry sites (IRESs), and identify combinations that result in high levels of circular mRNA and protein expression in cells. We show that these circular mRNAs can be packaged into virus-like particles (VLPs) thus enabling prolonged protein expression. Overall, these data describe a platform for synthesis of circular mRNAs and how these circular mRNAs can markedly enhance the ability of VLPs to function as a mRNA delivery tool.