Disclosed herein are engineered AAV VP capsid polypeptides with the ability to assemble into virus particles and having improved tissue tropism to, for example, CNS tissues. The capsids are engineered using the high throughput discovery system described herein. In certain embodiments, provided herein are recombinant adeno-associated virus (AAV) VP capsid polypeptides having at least one mutation in a residue corresponding to residue 581 to residue 589 in SEQ ID NO: 1.

The present invention relates to a polynucleotide comprising a Complement Factor I (CFI) nucleotide sequence encoding a CFI polypeptide or a fragment thereof. The invention further relates to a viral particle comprising a recombinant genome comprising the polynucleotide of the invention, and a composition comprising the polynucleotide or viral particle of the invention. The invention also relates to methods of using, and uses of, the polynucleotide, viral particle and/or composition of the invention. The invention also relates to methods of using, and uses of, a polynucleotide comprising a CFI nucleotide sequence, a viral particle comprising a recombinant genome comprising the polynucleotide, or a composition comprising the polynucleotide or viral particle.

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. The disclosed compositions and method entail making “reagent bundles” comprising many (hundreds of thousands to millions) clonal copies of an editing cassette and delivering or co-localizing the reagent bundles with live mammalian cells such that the editing cassettes edit the cells and the edited cells continue to grow.

The invention relates to methods for the simultaneous expression and delivery to mitochondria of two or more proteins using a single expression vector. Also described are the expression vectors and host cells comprising the vectors. Where the proteins are genome editing reagents, the invention also relates to the use of the expression vectors to alter levels of mitochondrial heteroplasmy and treat mitochondrial disorders.

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 invention provides an isolated chimeric virus comprising bocavirus capsid protein, e.g., an isolated chimeric virus comprising human bocavirus capsid protein, and a recombinant adeno-associated viral (AAV) genome, an isolated recombinant bocavirus (rBoV) comprising human bocavirus capsid protein and a recombinant Boy genome, and uses therefor, for example, in gene therapy for diseases in a mammal including diseases with aberrant expression of an endogenous gene product.

Compositions and methods are provided for preparation of concentrated stock solutions of AAV virions without aggregation. Formulations for AAV preparation and storage are high ionic strength solutions (e.g. μ˜500 mM) that are nonetheless isotonic with the intended target tissue. This combination of high ionic strength and modest osmolarity is achieved using salts of high valency, such as sodium citrate. AAV stock solutions up to 6.4×10.sup.13 vg/mL are possible using the formulations of the invention, with no aggregation being observed even after ten freeze-thaw cycles. The surfactant Pluronic® F68 may be added at 0.001% to prevent losses of virions to surfaces during handling. Virion preparations can also be treated with nucleases to eliminate small nucleic acid strands on virions surfaces that exacerbate aggregation.

Provided are mutant viral capsid cell libraries, individual cells of such libraries, systems, vectors, and methods for generating the cell libraries, and methods of use thereof to screen for mutant viral capsids with desired characteristics.

This invention relates to compositions of matter, methods, modules and instruments for automated mammalian cell growth and mammalian cell transduction followed by nucleic acid-guided nuclease editing in live mammalian 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.