Drug Discovery : Gene therapy


S1mplex: A New Tool for Precision Gene Editing

UW–Madison researchers have developed a modular RNA aptamer-streptavidin strategy, termed S1mplex, to ‘sharpen the scalpels’ used in genome surgery. In the new approach, CRISPR-Cas9 RNPs are complexed with a nucleic acid donor template, as well as other biotinylated molecules (e.g., quantum dots).

In human pluripotent stem cells, tailored S1mplexes increased the ratio of precisely edited to imprecisely edited alleles up to 18-fold higher than standard gene editing methods, and enriched cell populations containing multiplexed precise edits up to 42-fold.

These advances with versatile, preassembled reagents could greatly reduce the time and cost of in vitro/ex vivo gene editing applications in precision medicine and drug discovery, and aid in the development of increased and multiple dosing regimens for somatic gene editing in vivo.

Gene Editing Tool Uses New Enzyme

A UW–Madison researcher and collaborators have developed a method for RNA-directed DNA cleavage and gene editing using a new form of Cas9 enzyme. The new form, abbreviated NmCas9, comes from the Neisseria meningitides bacteria.

A CRISPR kit can be made containing RNA molecules that direct the new enzyme to bind to and cleave/nick a target sequence.

Charge-Dynamic Polymers for Delivering Anionic Compounds, Such as DNA, into Cells

UW-Madison researchers have developed polymers that allow temporal control over the dissociation of DNA from polymer/DNA interpolyelectrolyte complexes. The cationic polymers undergo dynamic changes in charge states (from cationic to less cationic) to trigger the “unpackaging” of anionic molecules from IPECs. The polymers possess cationic charge densities that result from the number, type, and position of functional groups attached to the backbone; specifically, cationic charge densities decrease when one or more of the functional groups is removed.

In one embodiment, side chain esters are introduced to linear poly(ethylene imine) (PEI) via conjugate addition chemistry. The PEI is then complexed with an anionic molecule such as DNA. When the pendant ester groups are hydrolyzed, the cationic charge density of the polymer is reduced, promoting the dissociation of the polymer/DNA complex and efficient release of DNA.

High Titer Recombinant Influenza Viruses for Vaccines and Gene Therapy

A UW-Madison researcher has developed an efficient technique and system for producing high titer influenza A virus in vertebrate cells in the absence of helper virus. The technology takes advantage of a reverse genetics system created by Dr. Kawaoka that allows efficient production of influenza virus for vaccines and gene therapy applications (see WARF reference number P03252US). In the technology described here, the inventor developed a new set of plasmids for use with the reverse genetics system. The plasmids contain cDNAs from a high titer influenza virus isolate; the promoter for RNA polymerase I or RNA polymerase II; and the terminator sequence for RNA polymerase I. When these constructs are transfected into host cells, the cells consistently generate high yields of infectious influenza particles.

Recombinant Influenza Vectors with a Pol II Promoter and Ribozymes for Vaccines and Gene Therapy

UW-Madison resarchers have developed an improved reverse genetics system for producing influenza virus in vertebrate cells in the absence of helper virus. The system starts with a set of plasmids containing viral genome cDNAs flanked by ribozymes. Each plasmid carries a cDNA for one of the eight influenza A viral RNA segments. On each plasmid, the cDNA sits between a polymerase II promoter and a poly-A addition signal at the 3-prime end. When the plasmids are transfected into a vertebrate cell, the host cell’s RNA polymerase II transcribes each construct into a capped viral RNA with a proper poly-A tail. The flanking ribozyme RNAs then undergo site-specific, self-catalyzed cleavage to precisely trim each end of the viral RNA. Next, viral polymerase, which is provided by a protein expression plasmid, acts upon the viral RNAs, resulting in replication and mRNA synthesis. This system does not require a helper virus and allows the creation of transfectants with mutations in any gene segment.

Methods for Engineering Influenza Viruses to Carry Defined Mutations

A UW-Madison reseasrcher has developed a method of preparing viruses with defined mutations. The method uses a reverse genetics system created by Dr. Kawaoka (see WARF reference number P03252US), which consists of plasmids containing the promoter for RNA polymerase I or RNA polymerase II; a cDNA for each of the influenza virus RNA segments; and the terminator for RNA polymerase I. These plasmids are transfected into cells along with protein expression plasmids to generate live virus.

The technology featured here allows mutations to be introduced into any of the cDNAs to generate viruses with defined mutations. For example, viruses lacking the NB protein -- an integral membrane glycoprotein that promotes efficient replication in vivo -- were created with this method. The NB knockout viruses replicated as efficiently as wild type virus in cell culture, but were attenuated in mice.