Disclosed herein are nucleic acid constructs that can be used to build genetic circuits for producing antibodies comprising split toxins. Also disclosed herein are methods of producing platelets comprising the antibodies. The platelets produced by the methods disclosed herein can be used to target circulating tumor cells.

Provided are anti-glypican-3 (GPC3) antibodies or antigen binding fragments thereof, and a chimeric antigen receptor (CAR) that binds glypican-3 (GPC3) containing an anti-GPC3 antibody in an extracellular domain, a transmembrane domain, and an intracellular signaling domain. Immune effector cells transduced with the disclosed CAR constructs can be used for cancer immunotherapy.

The present disclosure provides compositions and methods for conducting prime editing of a target DNA molecule (e.g., a genome) that enables the incorporation of a nucleotide change and/or targeted mutagenesis. The nucleotide change can include a single-nucleotide change (e.g., any transition or any transversion), an insertion of one or more nucleotides, or a deletion of one or more nucleotides. More in particular, the disclosure provides fusion proteins comprising nucleic acid programmable DNA binding proteins (napDNAbp) and a polymerase (e.g., reverse transcriptase), which is guided to a specific DNA sequence by a modified guide RNA, named an PEgRNA. The PEgRNA has been altered (relative to a standard guide RNA) to comprise an extended portion that provides a DNA synthesis template sequence which encodes a single strand DNA flap, which is homologous to a strand of the targeted endogenous DNA sequence to be edited, but which contains the desired one or more nucleotide changes and which, following synthesis by the polymerase (e.g., reverse transcriptase), becomes incorporated into the target DNA molecule. Also disclosed herein are various methods that leverage prime editing, including treating trinucleotide repeat contraction diseases, installing targeted peptide tags, treating prion disease through the installation of protection mutations, manipulating RNA-encoding genes for the installation of RNA tags for controlling the function and expression of RNA, using prime editing to construct sophisticated gene libraries, using prime editing to insert immunoepitopes into proteins, use of prime editing to insert inducible dimerization domains into protein targets, and delivery methods, among others.

The present disclosure relates to methods of protein purification by attaching an intein-C fragment to a target protein, passing a sample containing the intein-C tagged protein over a chromatographic resin carrying an intein-N fragment so as to create an intein-N intein-C complex, releasing the target protein from the intein-C fragment, and regenerating the column under conditions that disrupt the intein-N intein-C complex while preserving column functionality for multiple reuses.

Methods and constructs for the cleavage of polypeptides at one or more specific positions within the polypeptide are provided.

The present invention relates to methods of use of engineered split inteins, as well as their combination with degradation signals (destabilizing domains, degrons) to reconstitute large genes for gene therapy.

The disclosure provides methods, base editors, vectors encoding base editors and cognate gRNAs, and compositions and kits comprise said components, for installing nucleobase edits to the SMN2 locus to increase the activity and/or amount and/or stability of SMN2 protein in a cell, thereby treating Spinal Muscular Atrophy. In certain aspect, the disclosure provides compositions and methods to edit C840T of exon 7 of the SMN2 gene, or installing another one or more nucleobase edits which have the effect of removing or inactivating a degron, such as the C-terminal portion of the region encoded by exon 6 or the 4-amino acid region encoded by exon 8 (i.e., the EMLA (SEQ ID NO: 466)-tail) so as to remove or limit their degron activity to reduce, mitigate, or eliminate the intracellular degradation of the SMN2 protein.

The invention relates to transgene expressing Newcastle Disease Viruses (NDV), which have been demonstrated to possess significant oncolytic activity against mammalian cancers. The invention provides novel oncolytic viruses through the use of genetic engineering, including the transfer of foreign genes or parts thereof, such as genes encoding Ipilimumab, interleukin-12 or NS1. The present invention also provides nucleic acids encoding a reverse genetically engineered (rg-)NDV comprising one or more of these foreign genes and having a mutation in the HN gene, said mutation allowing replication of said rgNDV in a cancer cell to a higher level than replication of an otherwise identical rgNDV not having said mutation in the HN gene.

The present disclosure provides compositions and methods for conducting prime editing of a target DNA molecule (e.g., a genome) that enables the incorporation of a nucleotide change and/or targeted mutagenesis. The nucleotide change can include a single-nucleotide change (e.g., any transition or any transversion), an insertion of one or more nucleotides, or a deletion of one or more nucleotides. More in particular, the disclosure provides fusion proteins comprising nucleic acid programmable DNA binding proteins (napDNAbp) and a polymerase (e.g., reverse transcriptase), which is guided to a specific DNA sequence by a modified guide RNA, named an PEgRNA. The PEgRNA has been altered (relative to a standard guide RNA) to comprise an extended portion that provides a DNA synthesis template sequence which encodes a single strand DNA flap, which is homologous to a strand of the targeted endogenous DNA sequence to be edited, but which contains the desired one or more nucleotide changes and which, following synthesis by the polymerase (e.g., reverse transcriptase), becomes incorporated into the target DNA molecule. Also disclosed herein are various methods that leverage prime editing, including treating trinucleotide repeat contraction diseases, installing targeted peptide tags, treating prion disease through the installation of protection mutations, manipulating RNA-encoding genes for the installation of RNA tags for controlling the function and expression of RNA, using prime editing to construct sophisticated gene libraries, using prime editing to insert immunoepitopes into proteins, use of prime editing to insert inducible dimerization domains into protein targets, and delivery methods, among others.

A cassette sequence for the transformation of a host cell includes at least: a first nucleotide sequence encoding a peptide or protein of interest to be produced by the host cell. The first sequence is linked to a second nucleotide sequence providing resistance to a toxin or encoding an antitoxin peptide to the toxin. The nucleotide sequences are organized in such a way that production of the peptide encoded by the second nucleotide sequence(s) is translationally coupled to production of the peptide encoded by the first nucleotide sequence.