The present invention generally relates to improved methods of assembly of two or more DNA fragments, methods of rapid ligation-independent cloning, and kits for rapid ligation-independent cloning and their uses.

The present invention generally relates to improved methods of assembly of two or more DNA fragments, methods of rapid ligation-independent cloning, and kits for rapid ligation-independent cloning and their uses.

The present invention relates to antibodies or antigen binding fragment thereof that binds specifically to IGFBP3. The antibody inhibits or reduces the binding of IGFBP3 to the TMEM219 receptor. The invention also relates to methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.

The present invention relates to antibodies or antigen binding fragment thereof that binds specifically to IGFBP3. The antibody inhibits or reduces the binding of IGFBP3 to the TMEM219 receptor. The invention also relates to methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.

CRISPR RNA-guided nucleases are routinely used for sequence-specific manipulation of DNA. While CRISPR-based DNA editing has become routine, analogous methods for editing RNA have yet to be established. Here we repurpose the type III-A CRISPR RNA-guided nuclease for sequence-specific cleavage of the SARS-CoV-2 genome. The type III cleavage reaction is performed in vitro using purified viral RNA, resulting in sequence-specific excision of 6, 12, 18 or 24 nucleotides. Ligation of the cleavage products is facilitated by a DNA splint that bridges the excision and RNA ligase is used to link the RNA products before transfection into mammalian cells. The SARS-CoV-2 RNA is infectious and standard plaque assays are used to recover viral clones. Collectively, this work demonstrates how type III CRISPR systems can be repurposed for sequence-specific editing of RNA viruses including SARS-CoV-2 and more generally for gene therapy.

CRISPR RNA-guided nucleases are routinely used for sequence-specific manipulation of DNA. While CRISPR-based DNA editing has become routine, analogous methods for editing RNA have yet to be established. Here we repurpose the type III-A CRISPR RNA-guided nuclease for sequence-specific cleavage of the SARS-CoV-2 genome. The type III cleavage reaction is performed in vitro using purified viral RNA, resulting in sequence-specific excision of 6, 12, 18 or 24 nucleotides. Ligation of the cleavage products is facilitated by a DNA splint that bridges the excision and RNA ligase is used to link the RNA products before transfection into mammalian cells. The SARS-CoV-2 RNA is infectious and standard plaque assays are used to recover viral clones. Collectively, this work demonstrates how type III CRISPR systems can be repurposed for sequence-specific editing of RNA viruses including SARS-CoV-2 and more generally for gene therapy.

The present disclosure relates to gene therapy targeting GluK2 subunit that can be used to inhibit epileptiform discharges. Short interfering RNA sequences against the human Grik2 gene sequence are described which are efficient in decreasing the expression of GluK2-containing KARs in neurons engineered to express the equivalent shRNA or miRNA. Using a tissue culture model of TLE, the examples remarkably demonstrate that viral expression of shRNA or miRNA inhibits the frequency of epileptiform discharges. Therefore, RNA therapeutics aimed at decreasing the expression of GluK2-containing KARs in neurons can remarkably prevent spontaneous epileptiform discharges in TLE. In particular, the present disclosure relates to a recombinant antisense oligonucleotide that targets a Grik2 mRNA. The present disclosure also relates to a method for treating epilepsy in a subject in need thereof, wherein the method comprises: administering an effective amount of a vector comprising an oligonucleotide encoding an inhibitory RNA that binds (e.g., hybridizes) specifically to Grik2 mRNA and inhibits expression of Grik2 in the subject.

The present disclosure relates to gene therapy targeting GluK2 subunit that can be used to inhibit epileptiform discharges. Short interfering RNA sequences against the human Grik2 gene sequence are described which are efficient in decreasing the expression of GluK2-containing KARs in neurons engineered to express the equivalent shRNA or miRNA. Using a tissue culture model of TLE, the examples remarkably demonstrate that viral expression of shRNA or miRNA inhibits the frequency of epileptiform discharges. Therefore, RNA therapeutics aimed at decreasing the expression of GluK2-containing KARs in neurons can remarkably prevent spontaneous epileptiform discharges in TLE. In particular, the present disclosure relates to a recombinant antisense oligonucleotide that targets a Grik2 mRNA. The present disclosure also relates to a method for treating epilepsy in a subject in need thereof, wherein the method comprises: administering an effective amount of a vector comprising an oligonucleotide encoding an inhibitory RNA that binds (e.g., hybridizes) specifically to Grik2 mRNA and inhibits expression of Grik2 in the subject.

The present invention relates to, inter alia, an engineered cell (e.g., iPSC, IPS-derived NK, or NK cell) comprising a disrupted B2M gene and an inserted polynucleotide encoding one or more of SERPINB9, a fusion of IL15 and IL15Rα, and/or HLA-E. The engineered cell can further comprise a disrupted CIITA gene and an inserted polynucleotide encoding a CAR, wherein the CAR can be an anti-BCMA CAR or an anti-CD30 CAR. The engineered cell may further comprise a disrupted ADAM17 gene, a disrupted FAS gene, a disrupted CISH gene, and/or a disrupted REGNASE-1 gene. Methods for producing the engineered cells are also provided, and therapeutic uses of the engineered cells are also described. Guide RNA sequences targeting described target sequences are also described.

The present invention relates to, inter alia, an engineered cell (e.g., iPSC, IPS-derived NK, or NK cell) comprising a disrupted B2M gene and an inserted polynucleotide encoding one or more of SERPINB9, a fusion of IL15 and IL15Rα, and/or HLA-E. The engineered cell can further comprise a disrupted CIITA gene and an inserted polynucleotide encoding a CAR, wherein the CAR can be an anti-BCMA CAR or an anti-CD30 CAR. The engineered cell may further comprise a disrupted ADAM17 gene, a disrupted FAS gene, a disrupted CISH gene, and/or a disrupted REGNASE-1 gene. Methods for producing the engineered cells are also provided, and therapeutic uses of the engineered cells are also described. Guide RNA sequences targeting described target sequences are also described.