Leland Stanford Junior University

Poetic Genetics, LLC

Michele P Calos

Daniel V Palanker

Mark S Blumenkranz

Alexander B Vankov

Thomas W Chalberg, Jr

Ruby Yanru Tsai

Philip Huie, Jr

Birgitt Schüle

Simon Hippenmeyer

Bosiljka Tasic

Fangfang Zhu

Matthew Gamboa

Michael F Marmor

Alfonso P Farruggio

Christopher R Sclimenti

Methods for inserting a polynucleotide sequence into the genome of a human cell are provided. The present methods result in insertion of a polynucleotide sequence of interest into the H11 locus in the genome of a human cell. Also provided are nucleic acids that include sequences for integrating a polynucleotide sequence of interest into the H11 locus in the genome of a human cell. A transgenic human cell including site specific recombination sites at the H11 locus is also disclosed.

Site-specific integration of transgenes into human cells

The subject invention provides a unidirectional site-specific integration system for integrating a nucleic acid into the genome of a target cell. The provided systems include a (1) a mutant, unidirectional site specific integrase, which can be provided by an integrase vector encoding the mutant integrase and (2) a targeting vector that includes: (a) a nucleic acid to be integrated; and (b) a vector attachment site, where the targeting vector attachment site serves as a substrate for the mutant, unidirectional site-specific integrase. In using the subject systems for site-specific integration, the targeting vector and integrase are introduced into the target cell and the cell is maintained under conditions sufficient to provide for site-specific integration of the nucleic acid into the target cell genome via a recombination event mediated by the site-specific integrase. Also provided are kits that include the subject systems. The subjects systems, methods and kits find use in a variety of different applications, several representative ones of which are described in detail as well.

Methods of unidirectional, site-specific integration into a genome, compositions and kits for practicing the same

The subject invention provides a unidirectional site-specific integration system for integrating a nucleic acid into the genome of a target cell. The provided system includes a site-specific integrating expression cassette (INTEC) vector, consisting of (a) a polynucleotide of interest operably linked to a promoter, (b) a single recombination site, and (c) a hybrid recombination site. In using the subject systems for site-specific integration, the INTEC vector and integrase are introduced into the target cell and the cell is maintained under conditions sufficient to provide for site-specific integration of the nucleic acid into the target cell genome via a recombination event mediated by the site-specific recombinase. Also provided are kits that include the subject systems. The subjects systems, methods and kits find use in a variety of different applications, several representative ones of which are described in detail as well.

The present invention provides methods of site-specifically integrating a polynucleotide sequence of interest in a genome of a eucaryotic cell, as well as, enzymes, polypeptides, and a variety of vector constructs useful therefore. In the method, a targeting construct comprises, for example, (i) a first recombination site and a polynucleotide sequence of interest, and (ii) a site-specific recombinase, which are introduced into the cell. The genome of the cell comprises a second recombination site. Recombination between the first and second recombination sites is facilitated by the site-specific recombinase. The invention describes compositions, vectors, and methods of use thereof, for the generation of transgenic cells, tissues, plants, and animals. The compositions, vectors, and methods of the present invention are also useful in gene therapy techniques.

Methods and compositions for genomic modification

The present invention provides a method of treating an ocular disease in a subject. In a first step, a nucleic acid is introduced into cells or a tissue. The nucleic acid is introduced by electron avalanche transfection. With this technique, a high electric field induces a vapor bubble and plasma discharge between an electrode and the surrounding medium. The formation of a vapor bubble generates mechanical stress. Plasma discharge through the ionized vapor in the bubble enables connectivity between the electrode and the surrounding medium, so that mechanical stress and electric field are applied simultaneously, which results in permeabilization of the cells or tissue. This permeabilization in turn allows the nucleic acid to enter the cell or tissue. Cells or tissue containing the nucleic acid are then transplanted into an ocular region of the subject.

Ocular gene therapy using avalanche-mediated transfection

The present invention provides methods to site-specifically manipulate genomes by using hybrid recombinases. Hybrid recombinases comprise a modified catalytic domain from a unidirectional serine phage integrase, fused to a foreign DNA recognition domain.

Hybrid recombinases for genome manipulation

The present invention describes methods of identifying altered recombinases and compositions thereof, wherein at least one amino acid is different from a parent, wild-type recombinase and the altered recombinase has improved recombination efficiency towards wild-type and/or pseudo att site sequences relative to the parent, wild-type recombinase. The present invention also includes methods of modifying the genomes of cells using the altered recombinases, including methods of site-specifically integrating a polynucleotide sequence of interest in a genome of a eucaryotic cell.

Altered recombinases for genome modification

The present invention describes an expression vector useful for transfection of a selected mammalian host cell. The vector includes the following components: an Epstein Barr Virus (EBV) family of repeats; a copy of the EBV Nuclear Antigen-1 (EBNA-1) gene that can be functionally expressed in the host cell; a eucaryotic DNA fragment, which provides the ability of the vector to replicate in host cells; and an expression cassette which comprises a promoter functional in said host cell, a coding sequence having 5' and 3' ends, where said coding sequence is functionally linked to said promoter, where said 5' end is adjacent the promoter and said 3' end is adjacent transcription termination sequences. The vector of the present invention is useful in the transfection of mammalian cells, including rodent and human cells. The vector is stably retained and replicates in concert with genomic sequences of the host cell, that is, the vector is typically replicated once per cell cycle. The vector is useful in the follow methods involving mammalian cells: producing proteins, stable transformation, and gene therapy.

Autonomous replicaton system for mammalian cells

Mutagenic capability is determined by employing a shuttle vector having prokaryotic and eukaryotic origins, a prokaryotic marker an a gene capable of screening or selection in a prokaryote. The method involves introducing the vector into mammalian cells, exposing the cells to the candidate to be tested for mutagenicity for a time sufficient to allow lesions to occur, rescuing the vector by transforming into a prokaryotic host and screening for mutations of the gene.

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Woodside, CA, United States of America

Michele P Calos