Site News  |   Monitor Keywords  |   Monitor Archive  |   Organizer  |   Account Info  |

High-throughput methods for identifying gene function using lentiviral vectors

High-throughput methods for identifying gene function using lentiviral vectors description/claims

This application is a continuation of application Ser. No. 10/627,940, filed on Jul. 25, 2003 which is a continuation-in-part (“CIP”) of PCT/US02/02287, filed Jan. 25, 2002 and designating the United States, which claims benefit of priority to U.S. Provisional Patent Application 60/264,272, filed Jan. 25, 2001, both of which are hereby incorporated by reference in their entireties as if fully set forth.

The present invention is directed to methods, as well as compositions related thereto, for the efficient identification of one or more functionalities of a product encoded by a nucleic acid sequence. The methods utilize the abilities to over and/or under express the product in a cell, as well as the combination of these results, to permit the identification of at least one of the product's cellular or in vivo functionality.

The tremendous efforts at sequencing the genomes of human beings and other organisms has produced a vast amount of nucleic acid and protein sequence information for additional analysis. Much of the sequence information is now, or will be, the subject of both biochemical and functional characterization. The sequence information also serves as the raw material for “bioinformatics”, where the sequence itself is used in comparisons with other sequences for which the structure, function, or other characteristics have been previously identified. The great hope and expectation for these efforts is that with the identification of functionalities encoded by genetic sequences, additional therapeutic products and treatments can be developed for diseases in humans and other organisms.

The effort to identify functions encoded by genetic sequences has focused, at least initially, on sequences that encode actual gene products, or “genes”. Earlier approaches sought to clone and sequence only genes based on tools and strategies for using positional cloning to map and clone genes. While labor intensive, positional cloning has been successful in locating genes associated with various diseases. Initially, genetic mapping is performed based on large families of related individuals to locate a disease associate gene at the level of chromosomal location and in the range of centimorgans. Next, and with a significant increase in effort, the work becomes one of physically mapping the genes so that centimorgans are reduced to megabasepairs and then finally to particular nucleotides. Examples of successes with positional cloning include the identification of genes associated with cystic fibrosis and Huntington's disease.

Other approaches to the isolation of genes include exon trapping (Buckler et al. (1991) P.N.A.S. 88:4005-4009) and direct selection (Morgan et al. (1992) N.A.R. 20:5173-5179). These methods identify potential genes in large genomic regions which are then sequenced and used in confirming the genes as actually expressed. In some cases, cells that normally express the potential gene are unknown, and it remains necessary to confirm the expression of the genes and identify the functionality of the encoded product.

An initial advantage available with positional cloning over the above two methods is that there is no need for knowledge concerning the functional or physiological role of the gene product of the identified gene. The identification is made based on following a phenotypic trait followed by studying genetic segregation of a particular sequence with the trait. But after identification, there may still be difficulties in determining the functional role of the gene product for the design of appropriate therapies. Without knowing the functional role of the encoded product, it remains difficult, for example, to identify suitable agents to use as pharmaceuticals to appropriately target the gene product. Additionally, it remains unknown how the identified gene is involved in the progression from onset and progression to the later stages of the disease.

A more recent approach to the isolation of genes has been based on massive sequencing efforts designed to identify all expressed sequences in a genome. Completion of such efforts in the human and Drosophila genomes, as well as some microorganisms, have been recently reported. But with the production of such large amounts of sequence information, the need for a rapid and efficient means for identifying the functionality of encoded gene products increases further. This need has led to intensive commercial and industrial activity for additional methods to identify gene function.

One means for identifying function is through bioinformatics, which seeks to determine functionality based on similarities between a new sequence and other sequences for which the structure, function, or other characteristics have been previously identified. Bioinformatics is most often performed with computer programs and thus have been termed to occur “in silico”. One drawback of bioinformatics, however, is that it only provides a starting point for possibly validating a postulated functionality of a gene sequence. Until a new sequence is actually expressed and characterized within a living cell or organism, the supposed functionality remains a hypothesis to be proven.

An approach to validate an assigned gene function is via the use of small animal models. For example, transgenic mice have been used for the overexpression of gene sequences in attempts to identify the encoded functionality. Gene sequences have also been used in the production of “knockout” mice where the endogenous mouse sequence is no longer expressed. But the time and cost of transgenic approaches have limited their usefulness to studies of only a few sequences at a time.

Another approach has been to make use of cell cultures to overexpress a gene sequence of interest. Unfortunately, there is no rapid and efficient means for reliably producing a “knockout” cell where the endogenous cellular sequence is not expressed or overexpressed. Overexpression methods are, however, limited by the vector system used to deliver and express the gene. As an initial matter, known vector systems limit the number of cells that are transfected with the gene. For example, plasmid vectors have low transfection efficiencies and thus require the use of a selectable marker to isolate transfected cells. But the expression of a marker gene from the plasmid vector tends to skew the phenotype detected because the gene of interest is not the only gene being overexpressed in the cell. Stated differently, expression of the gene of interest is not the only initial perturbation occurring in the cell. As such, the determination of gene function may be significantly mistaken due to skewing by expression of the marker gene. The same selectable marker mediated skewing is seen with some viral vectors, such as onco-retroviral vectors.

Higher transfection efficiencies are available from other viral vectors, such as adenovirus based vectors, but these vectors often fail to provide stable expression of the gene of interest. More importantly, such vectors often have large numbers of their own genes to express or suffer the risk of contamination due to co-infection by helper virus. The expression of vector and/or helper virus genes again perturbs the intracellular environment and skews the detected phenotype and thus affects the determination of gene function.

An additional limitation on the use of vector based overexpression is found with the uncertainty as to what resultant phenotype should be, or can be, detected in the transfected cell. Moreover, such methods rarely use primary cells but instead use cell lines or diseased cells where any identified gene function remains suspect because of the abnormal cellular environment.

Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.

The present invention provides compositions and methods to increase the ability to identify one or more functions of products encoded by unidentified gene sequences or to further identify or confirm one or more functions of known gene sequences. Therefore, and in one aspect, the invention provides a lentiviral vector capable of high transduction in primary cells, preferably without altering the overall gene expression profile of the cell, except for the expression of a specific payload encoding, or targeted to, one or more gene sequences under investigation. Gene expression profile refers to the levels of expression, at the RNA and/or protein levels, of coding sequences in a cell.

The present invention thus provides a clear validating system for the determination of gene function, where the cellular effects of overexpression may be compared to and correlated with those of inhibition. The invention may be applied, as a non-limiting, but important example of a large scale gene chip experiment where the background level(s) of gene expression is a significant difficulty to data interpretation. The (cellular or endogenous) genes identified or confirmed to be affected by increased and/or decreased expression of a gene sequence of interest can be placed in a matrix for analysis to describe the function of the gene under investigation.

The present invention provides for the determination of one or more functionalities of a given unidentified or known gene sequence of interest by at least two means. First, the gene sequence, or one or more portions thereof, is inserted in a vector and introduced into a cell for expression of the encoded gene product. The level of expression can of course be attenuated, but preferably, the sequence is overexpressed. After expression occurs, changes in the expression, composition, or form of endogenous cellular factors, in comparison to normal cells without said vector, are detected and analyzed. This permits the identification of what cellular factors are affected by the sequence being expressed or overexpressed. Without limiting the scope of the invention, the actual effect on the cellular factor may include that of changes in its level of expression (e.g. at the protein and/or RNA levels), changes in its amino acid composition (e.g. number and type of subunits and/or splice variants), and changes in its state of post-translational modification (e.g. phosphorylation and/or glycosylation and/or lipid modification) or location (e.g. subcellular location as well as being soluble, membrane associated, or by insertion of at least one portion of the factor into the hydrophobic portion of a membrane). Cellular factors include those with one or more identified function as well as those for which a function has yet to be identified.

Second, expression of the unidentified or known gene sequence is inhibited or terminated in a cell. Without limiting the scope of the invention, the inhibition may be by use of all or part of the gene sequence to recombine with the endogenous copy or copies of the sequence in said cell to terminate its expression. Alternatively, the gene sequence, or one or more portions thereof, maybe inserted in an antisense orientation in a vector. The expression of the sequence, or portion thereof, may be regulated such that it is expressed only when desired to produce an antisense nucleic acid.

• Provisional Patent
• Utility Patent

• What Is a Patent?
• What Is a Trademark or Servicemark?
• What Is a Copyright?
• Patent Laws