This invention relates to compositions of matter, methods, modules and automated, end-to-end closed instruments for automated mammalian cell growth, reagent bundle creation and mammalian cell transfection followed by nucleic acid-guided nuclease editing in live mammalian cells.

Provided are vaccines, polypeptides and polynucleotides based on mutant CALR and JAK2 sequences, vectors, host cells, viruses, and methods of making and using them. The disclosure also provides methods of inducing an immune response and methods of treating, preventing, reducing a risk of onset or delaying the onset of a clinical condition characterized by an expression of JAK2V617F or CALR exon 9 mutant, or both JAK2V617F and CALR exon 9 mutant, wherein the method comprises a plurality of administrations of any of the compositions comprising polynucleotides, polypeptides or vectors disclosed herein.

Provided is a method of inserting a polynucleotide sequence into a genome of a cell. The method comprises: generating a double-strand break at a target site of the genome; and introducing into the cell a virus. The virus comprises a nucleic acid comprising the polynucleotide sequence to be inserted or the complementary sequence thereof. The nucleic acid does not comprise a homologous arm or comprises very short (5˜25 bp) homologous arms corresponding to the target site. Also provided herein is a composition for inserting a polynucleotide sequence into a genome of a cell. The composition comprises a site-specific nuclease capable of generating a DNA double-strand break at a target site of the genome and a virus comprising a nucleic acid comprising the polynucleotide sequence or the complementary sequence thereof.

The present invention provides compositions and methods of use pertaining to rAAV-mediated delivery of therapeutically effective molecules for treatment of diseases such as Pompe disease. These compositions in combination with various routes and methods of administration result in targeted expression of therapeutic molecules in specific organs, tissues and cells.

The current invention relates to nucleic acid impurities in a composition comprising a parvoviral vector. In particular, the current invention shows that DNA impurities are not randomly encapsulated within a parvoviral virion. The invention therefore relates to a method for identifying and quantifying a nucleic acid impurity in a composition comprising a parvoviral vector. Finally, the current invention relates to method of determining whether a composition comprising a parvoviral vector is regarded as clinically pure.

The present invention is directed to methods and compositions for treating or attenuating age-related symptoms or diseases in a cell, a tissue, an organ, or an organism by administration of therapeutically effective amounts of VEGF stimulating-, VEGFR stimulating-compound, or any combination thereof.

The AAV compositions comprising an AAV and a buffer. These AAV compositions are appropriate for us in intracerebroventricular (ICV) and intraparenchymal (IPA) administration, as well as other routes of administration and can be provided in frozen, liquid or potentially freeze dried (lyophilized) state.

Nucleic acid-guided nuclease editing in mammalian cells may include passaging mammalian cells, in an automated closed cell editing instrument, into smaller aggregates when the aggregates exceed 50-300 microns in size. A library of viral particles may be delivered to the mammalian cells at a multiplicity of infection such that each mammalian cell receives one or no viral particle. The library may include viral vectors with an editing cassette including a pair of gRNA coding sequence and donor DNA. Conditions may be provided to allow a viral vector of the viral vectors to integrate into the mammalian cells. Enriching for mammalian cells may be done with an integrated viral vector. A nucleic acid-guided nuclease or nuclease fusion or a coding sequence for a nucleic acid-guided nuclease or nuclease fusion may be delivered to the enriched mammalian cells and conditions may be provided to allow editing in the mammalian cells.

The present invention provides isolated promoters, transgene expression cassettes, vectors, kits, and methods for treatment of age-related macular generation and other genetic diseases that affect the cone cells of the retina.

Methods for preparing highly purified AAV vector formulations are provided. The highly pure AAV formulations described herein are superior for clinical use.