A potent short hairpin RNA (shRNA734) directed to human Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) improves the rate of gene-modified stem cell engraftment by a conditioning and in vivo selection strategy to confer resistance to a clinically available guanine analog antimetabolite, 6TG, for efficient positive selection of gene-modified stem cells. Uses for polynucleotides comprising the shRNA734 include methods for knocking down HPRT in a cell, for conferring resistance to a guanine analog antimetabolite in a cell, for producing selectable genetically modified cells, for selecting cells genetically modified with a gene of interest from a plurality of cells, for removing cells genetically modified with a gene of interest from a plurality of cells, and for treating a subject infected with HIV.

Compositions, recombinant viral vectors, recombinant viruses, and nanoparticles for treating a subject having a spinal cord injury include a therapeutically effective amount of a nucleic acid sequence encoding pre-microRNA-7 (pre-miR-7). Methods of using these compositions, recombinant viral vectors, recombinant viruses, and nanoparticles are also described herein. These compositions, recombinant viral vectors, recombinant viruses, and nanoparticles and methods of use provide novel therapies for SCI based on the discovery that miR-7 expression provides neuroprotection and recovery of locomotor function in subjects having SCI.

The invention pertains to an inducible CRISPR system for controlling expression of a CRISPR complex with an inducible fusion promoter. One embodiment of the invention provides HIV LTR-minimal Drosophila hsp70 fusion promoter that can be used for inducible co-expression of gRNA and Cas9 in HIV-infected cells to target cellular cofactors such as Cyclin T1. A single introduction of such embodiment leads to sustained suppression of HIV replication in stringent, chronically infected HeLa-CD4 cell lines as well as in T-cell lines. In another embodiment, the invention further relates to enhancement of HIV suppression by incorporating cis-acting ribozymes immediately upstream of the gRNA in the inducible CRISPR system construct. The inducible fusion promoter is adaptable for other tissue- or cell-type specific expression of the inducible CRISPR system.

The present application discloses a lentiviral transfer system which includes: (i) a self-inactivating transfer vector comprising: multiple gene units, wherein each gene unit includes a heterologous nucleic acid sequence operably linked to a regulatory nucleic acid sequence; and (ii) a helper construct which lacks a 5′ LTR, wherein the 5′ LTR has been replaced with a heterologous promoter, in which the helper construct further comprises: a lentiviral env nucleic acid sequence containing a deletion, wherein the deleted env nucleic acid sequence does not produce functional env protein; and a packaging signal contains a deletion, wherein the deleted packaging signal is nonfunctional.

There is disclosed a trans-acting functional nucleic acid molecule comprising a eukaryotic target binding sequence comprising a sequence reverse complementary to a target mRNA sequence for which protein translation is to be enhanced, and a regulatory sequence comprising an internal ribosome entry site (IRES) sequence or an internal ribosome entry site (IRES) derived sequence and enhancing translation of the target mRNA sequence, wherein the regulatory sequence is located 3′ of the target binding sequence.

The present invention provides methods of generating a recombinant AAV vector with reduced immunogenicity, comprising: providing eukaryotic cells with a nucleic acid comprising a sequence of interest that is flanked by AAV inverted terminal repeats, wherein the nucleic acid comprises CpG dinucleotide sites, wherein at least a portion of the CpG dinucleotide sites are methylated, wherein the eukaryotic cell expresses one or more other components necessary to achieve recombinant AAV biosynthesis, whereby the recombinant AAV vector is generated by the eukaryotic cell, wherein the generated recombinant AAV vector comprises nucleic acid wherein at least a portion of the CpG dinucleotide sites are methylated.

The invention pertains to an inducible CRISPR system for controlling expression of a CRISPR complex with an inducible fusion promoter. One embodiment of the invention provides HIV LTR-minimal Drosophila hsp70 fusion promoter that can be used for inducible co-expression of gRNA and Cas9 in HIV-infected cells to target cellular cofactors such as Cyclin T1. A single introduction of such embodiment leads to sustained suppression of HIV replication in stringent, chronically infected HeLa-CD4 cell lines as well as in T-cell lines. In another embodiment, the invention further relates to enhancement of HIV suppression by incorporating cis-acting ribozymes immediately upstream of the gRNA in the inducible CRISPR system construct. The inducible fusion promoter is adaptable for other tissue- or cell-type specific expression of the inducible CRISPR system.

The present invention provides a method and a pharmaceutical composition for the treatment of the NDO comprising the viral expression vector carrying a transcription cassette that harbors transgene(s) inhibiting/silencing neurotransmission or synaptic transmission of afferent neurons.

The present disclosure provides methods for generating induced cardiomyocytes by expression of selected microRNAs with MYOCD and ASCL1, or with MYOCD alone. Illustrative microRNAs include miR-133, miR-1, miR-19, and/or miR-20b. The present disclosure further provides gene-delivery vectors comprising one or more polynucleotides encoding a selected microRNA with YOCD, withMYOCD and ASCL1, with MYOCD-2A-ASCL1, or with ASCL1-2A-MYOCD. It further provides methods of using such compositions and vectors, or induced cardiomyocytes generated with these factors, for treating a heart condition.

A recombinant viral vector comprising an expression cassette which comprises a coding sequence for an shRNA inhibitor of vasohibin (VASH)-small vasohibin binding protein (SVBP) complex operably linked to regulatory sequences which direct expression thereof is provided. Further provided are compositions containing such viral vectors formulated for delivery to a human patient. Also provided are methods using these vectors and compositions for improving or stabilizing cardiac function.