Evaluation of Mono- and Bi-Functional GLOBE-Based Vectors for Therapy of ß-Thalassemia by HBBAS3 Gene Addition and Mutation-Specific RNA Interference.

Therapy via the gene addition of the anti-sickling ßAS3-globin transgene is potentially curative for all ß-hemoglobinopathies and therefore of particular clinical and commercial interest. This study investigates GLOBE-based lentiviral vectors (LVs) for ßAS3-globin addition and evaluates strategies for an increased ß-like globin expression without vector dose escalation. First, we report the development of a GLOBE-derived LV, GLV2-ßAS3, which, compared to its parental vector, adds anti-sickling action and a transcription-enhancing 848-bp transcription terminator element, retains high vector titers and allows for superior ß-like globin expression in primary patient-derived hematopoietic stem and progenitor cells (HSPCs). Second, prompted by our previous correction of HBBIVSI-110(G>A) thalassemia based on RNApol(III)-driven shRNAs in mono- and combination therapy, we analyzed a series of novel LVs for the RNApol(II)-driven constitutive or late-erythroid expression of HBBIVSI-110(G>A)-specific miRNA30-embedded shRNAs (shRNAmiR). This included bifunctional LVs, allowing for concurrent ßAS3-globin expression. LVs were initially compared for their ability to achieve high ß-like globin expression in HBBIVSI-110(G>A)-transgenic cells, before the evaluation of shortlisted candidate LVs in HBBIVSI-110(G>A)-homozygous HSPCs. The latter revealed that ß-globin promoter-driven designs for monotherapy with HBBIVSI-110(G>A)-specific shRNAmiRs only marginally increased ß-globin levels compared to untransduced cells, whereas bifunctional LVs combining miR30-shRNA with ßAS3-globin expression showed disease correction similar to that achieved by the parental GLV2-ßAS3 vector. Our results establish the feasibility of high titers for LVs containing the full HBB transcription terminator, emphasize the importance of the HBB terminator for the high-level expression of HBB-like transgenes, qualify the therapeutic utility of late-erythroid HBBIVSI-110(G>A)-specific miR30-shRNA expression and highlight the exceptional potential of GLV2-ßAS3 for the treatment of severe ß-hemoglobinopathies.

miR-AB, a miRNA-based shRNA viral toolkit for multicolor-barcoded multiplex RNAi at a single-cell level.

Uncovering the functions of genes in a complex biological process is fundamental for systems biology. However, currently there is no simple and reliable experimental tool available to conduct loss-of-function experiments for multiple genes in every possible combination in a single experiment, which is vital for parsing the interactive role of multiple genes in a given phenotype. In this study, we develop miR-AB, a new microRNA-based shRNA (shRNAmir) backbone for simplified, cost-effective, and error-proof production of shRNAmirs. After verification of its potent RNAi efficiency in vitro and in vivo, miR-AB was integrated into a viral toolkit containing multiple eukaryotic promoters to enable its application in diverse cell types. We further engineer eight fluorescent proteins emitting wavelengths across the entire visible spectrum into this toolkit and use it to set up a multicolor-barcoded multiplex RNAi assay where multiple genes are strongly and reliably silenced both individually and combinatorially at a single-cell level.

Coordinated ß-globin expression and α2-globin reduction in a multiplex lentiviral gene therapy vector for ß-thalassemia.

A primary challenge in lentiviral gene therapy of ß-hemoglobinopathies is to maintain low vector copy numbers to avoid genotoxicity while being reliably therapeutic for all genotypes. We designed a high-titer lentiviral vector, LVß-shα2, that allows coordinated expression of the therapeutic ßA-T87Q-globin gene and of an intron-embedded miR-30-based short hairpin RNA (shRNA) selectively targeting the α2-globin mRNA. Our approach was guided by the knowledge that moderate reduction of α-globin chain synthesis ameliorates disease severity in ß-thalassemia. We demonstrate that LVß-shα2 reduces α2-globin mRNA expression in erythroid cells while keeping α1-globin mRNA levels unchanged and ßA-T87Q-globin gene expression identical to the parent vector. Compared with the first ßA-T87Q-globin lentiviral vector that has received conditional marketing authorization, BB305, LVß-shα2 shows 1.7-fold greater potency to improve α/ß ratios. It may thus result in greater therapeutic efficacy and reliability for the most severe types of ß-thalassemia and provide an improved benefit/risk ratio regardless of the ß-thalassemia genotype.

Runx2 silencing promotes adipogenesis via down-regulation of DLK1 in chondrogenic differentiating MSCs.

BACKGROUND: For cartilage regeneration, stem cells are a promising cell source; however, even the advances made in the differentiation of stem cells into precursor-differentiated cartilage cells have not been successful with respect to reprograming these cells to achieve complete differentiation and fully functioning cells until now. Previous findings suggest that Runx2 plays a major role in chondrocyte differentiation and maturation. Although targeting Runx2 has enhanced some chondrocyte properties, the adipogenic lineage shift has eventually occurred in these cells. The present study mainly aimed to reveal the mechanism of this adipogenesis. METHODS: To create inducible artificial shRNA-miR expressing vectors, the designed short hairpin RNAs (shRNAs) were inserted into the pri-mir-30 backbone, cloned into lentiviral pLVET-Tet-on, and transducted into mesenchymal stem cells (MSCs). Runx2 gene was silenced in MSCs either for 1 week or 4 weeks and cultured in the chondrogenic medium. At days 7, 14 and 28, cells were harvested, and chondrogenesis, adipogenesis and hypertrophic states were examined using histochemical staining and a real-time polymerase chain reaction assay. RESULTS: The results showed that the designed shRNA-miR effectively targeted Runx2 in mRNA and protein levels. Chondrogenic markers were up-regulated in constantly silenced Runx2 group; however, adipogenic markers and fat droplets appeared gradually. DLK1 gene was also significantly down-regulated in this group, and overexpression of DLK1 abrogated adipogenesis in the Runx2 targeted group. CONCLUSIONS: Based on these results, it can be concluded that DLK1 is responsible for the lineage shift in Runx2 targeted chondrogenic differentiating MSCs.

Preclinical Evaluation of a Novel Lentiviral Vector Driving Lineage-Specific BCL11A Knockdown for Sickle Cell Gene Therapy.

In this work we provide preclinical data to support initiation of a first-in-human trial for sickle cell disease (SCD) using an approach that relies on reversal of the developmental fetal-to-adult hemoglobin switch. Erythroid-specific knockdown of BCL11A via a lentiviral-encoded microRNA-adapted short hairpin RNA (shRNAmiR) leads to reactivation of the gamma-globin gene while simultaneously reducing expression of the pathogenic adult sickle ß-globin. We generated a refined lentiviral vector (LVV) BCH-BB694 that was developed to overcome poor vector titers observed in the manufacturing scale-up of the original research-grade LVV. Healthy or sickle cell donor CD34+ cells transduced with Good Manufacturing Practices (GMP)-grade BCH-BB694 LVV achieved high vector copy numbers (VCNs) >5 and gene marking of >80%, resulting in a 3- to 5-fold induction of fetal hemoglobin (HbF) compared with mock-transduced cells without affecting growth, differentiation, and engraftment of gene-modified cells in vitro or in vivo. In vitro immortalization assays, which are designed to measure vector-mediated genotoxicity, showed no increased immortalization compared with mock-transduced cells. Together these data demonstrate that BCH-BB694 LVV is non-toxic and efficacious in preclinical studies, and can be generated at a clinically relevant scale in a GMP setting at high titer to support clinical testing for the treatment of SCD.

Stable STIM1 Knockdown in Self-Renewing Human Neural Precursors Promotes Premature Neural Differentiation.

Ca2+ signaling plays a significant role in the development of the vertebrate nervous system where it regulates neurite growth as well as synapse and neurotransmitter specification. Elucidating the role of Ca2+ signaling in mammalian neuronal development has been largely restricted to either small animal models or primary cultures. Here we derived human neural precursor cells (NPCs) from human embryonic stem cells to understand the functional significance of a less understood arm of calcium signaling, Store-operated Ca2+ entry or SOCE, in neuronal development. Human NPCs exhibited robust SOCE, which was significantly attenuated by expression of a stable shRNA-miR targeted toward the SOCE molecule, STIM1. Along with the plasma membrane channel Orai, STIM is an essential component of SOCE in many cell types, where it regulates gene expression. Therefore, we measured global gene expression in human NPCs with and without STIM1 knockdown. Interestingly, pathways down-regulated through STIM1 knockdown were related to cell proliferation and DNA replication processes, whereas post-synaptic signaling was identified as an up-regulated process. To understand the functional significance of these gene expression changes we measured the self-renewal capacity of NPCs with STIM1 knockdown. The STIM1 knockdown NPCs demonstrated significantly reduced neurosphere size and number as well as precocious spontaneous differentiation toward the neuronal lineage, as compared to control cells. These findings demonstrate that STIM1 mediated SOCE in human NPCs regulates gene expression changes, that in vivo are likely to physiologically modulate the self-renewal and differentiation of NPCs.

Efficient Gene Knockdowns in Mouse Embryonic Stem Cells Using MicroRNA-Based shRNAs.

RNA interference (RNAi) is a powerful gene knockdown technology that has been applied for functional genetic loss-of-function studies in many model eukaryotic systems, including embryonic stem cells (ESCs). Application of RNAi in ESCs allows for dissection of mechanisms by which ESCs self-renew and maintain pluripotency and also for specifying particular cell types needed for cell replacement therapies. Potent RNAi response can be induced by expression of a microRNA-embedded short-hairpin RNA (shRNAmir) cassette that is integrated in the genome by virus infection or site-specific recombination at a defined locus. In this chapter, I will provide detailed protocols to perform shRNAmir-mediated RNAi studies in mouse ESCs using retrovirus infection and loxP site-directed recombination for efficient constitutive and inducible gene knockdown, respectively.

Lentiviral vector-encoded microRNA-based shRNA-mediated gene knockdown of N-methyl-D-aspartate receptors in skin reduces pain.

BACKGROUND AND PURPOSE: RNA polymerase II promoters that drive the expression of rationally designed primary microRNA-based shRNA, for example, shRNAmir, can produce more potent gene knockdown than RNA polymerase III promoters. Antagonists of peripheral N methyl-D-aspartate (NMDA) receptors that do not interfere with central glutamate processing would prevent the development of adverse central nervous system effects. Thus, in this study, we examined the effects of gene silencing and antinociception on formalin- and Complete Freund's adjuvant (CFA)-induced pain in rats by subcutaneously injecting a lentiviral vector encoding a shRNAmir that targets the NR1 subunit of the NMDA receptor. METHODS: Rats received intradermal injections of different doses of NR1 shRNAmir at different time points before injection of formalin. Pain behavior was assessed by monitoring the paw flinch response, paw withdrawal threshold, and thermal withdrawal latency. We then analyzed NR1 messenger RNA and protein expression in skin and the L5 dorsal root ganglion (DRG). RESULTS: We found that intradermal injection of 1, 5, and 10 µg of shRNAmir significantly inhibited flinch responses (p < .05). Administration of 5 µg of shRNAmir resulted in the attenuation of CFA-induced mechanical allodynia, but did not affect the time spent on the rotarod. Real-time polymerase chain reaction and western blotting revealed that NR1 mRNA and protein levels were significantly lower in all NR1 shRNAmir1 groups than in controls (p < .05). There was a significant reduction in the percentage of NR1- and pERK-positive neurons in the DRG ipsilateral to shRNAmir treated paws (p < .05). The effect of antinociception and inhibition of NR1 expression by NR1 shRNAmir was evident on day 3 and persisted for 7 days after injection of 5 µg of vector. CONCLUSION: Peripheral administration of the vector-encoded NR1 shRNAmir is a promising therapy for persistent inflammatory pain.

Recent advances in RNAi-based strategies for therapy and prevention of HIV-1/AIDS.

RNA interference (RNAi) provides a powerful tool to silence specific gene expression and has been widely used to suppress host factors such as CCR5 and/or viral genes involved in HIV-1 replication. Newer nuclease-based gene-editing technologies, such as zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, also provide powerful tools to ablate specific genes. Because of differences in co-receptor usage and the high mutability of the HIV-1 genome, a combination of host factors and viral genes needs to be suppressed for effective prevention and treatment of HIV-1 infection. Whereas the continued presence of small interfering/short hairpin RNA (si/shRNA) mediators is needed for RNAi to be effective, the continued expression of nucleases in the gene-editing systems is undesirable. Thus, RNAi provides the only practical way for expression of multiple silencers in infected and uninfected cells, which is needed for effective prevention/treatment of infection. There have been several advances in the RNAi field in terms of si/shRNA design, targeted delivery to HIV-1 susceptible cells, and testing for efficacy in preclinical humanized mouse models. Here, we comprehensively review the latest advances in RNAi technology towards prevention and treatment of HIV-1.