News Column

Patent Application Titled "Optimization of Vectors for Effective Delivery and Expression of Genetic Content" Published Online

August 21, 2014



By a News Reporter-Staff News Editor at Gene Therapy Weekly -- According to news reporting originating from Washington, D.C., by NewsRx journalists, a patent application by the inventor Wakefield, John (Birmingham, AL), filed on October 2, 2012, was made available online on August 7, 2014 (see also Thermo Fisher Scientific Biosciences Inc.).

The assignee for this patent application is Thermo Fisher Scientific Biosciences Inc.

Reporters obtained the following quote from the background information supplied by the inventors: "Gene therapy approaches rely on the efficient transfer of genes to the desired target cells. In order to increase efficiency, researchers have explored ways to use both viral and non-viral vectors.

"One of the areas that researchers have explored with great interest is the use of lentiviral vectors. Lentiviral vectors make use of lentiviral polynucleotide sequences and are a subclass of retroviral vectors. However, unlike other retroviruses, lentiviruses are able to integrate into the genome of non-dividing cells. After entry into a cell, the viral genome, which is in the form of single-stranded RNA, is reverse transcribed to generate a double-stranded DNA, which is then inserted into the host genome. Because lentiviral vectors can cause their sequences to be integrated into non-dividing cells, they provide particularly promising leads for gene therapy.

"Another area of interest for researchers is RNA inference, also referred to as RNAi, which involves the phenomenon of gene silencing following the introduction of double-stranded RNA (dsRNA) into cells. In mammalian cells, in order to avoid an interferon response, RNAi is conducted by using short interfering RNA, also known as siRNA, which may comprise two separate strands of RNA or a hairpin structures also known as shRNA, that may be processed by a cell into a short duplex that contains two different strands.

"Lentiviruses have recently been viewed and tried as potential means of efficiently introducing shRNA into cells. When introduced into cells under conditions that permit the lentiviruses to act in their intended manners, these vectors have demonstrated the potential to be important tools for gene therapy.

"As persons of ordinary skill in the art will recognize, lentiviruses, like other vectors, are often created and introduced into cells with internal promoter regions, which serve the function of providing control points for transcription. However, promoter activity can vary widely across cell lines and cell types. Inefficient promoter activity can result in poor expression, which can often be misinterpreted as unsuccessful delivery, unsuccessful genomic integration or in the case of shRNA-containing vectors, poor gene silencing efficiency.

"Thus, there is a need to provide better options for permitting persons of ordinary skill in the art to select elements of their lentiviral vector constructs with greater likelihood that the constructs will effectively introduce the desired genetic element into any cell or organism and express the desired genetic elements in the cell or organism."

In addition to obtaining background information on this patent application, NewsRx editors also obtained the inventor's summary information for this patent application: "The present invention is directed to plates, kits, methods, and vectors that facilitate the design of efficient genetic constructs and the use of these constructs. Through various embodiments of the present invention, there is an ability to assess promoter activity in cells of interest prior to conducting a gene silencing or expression experiment of therapy. Advantageously, some of these embodiments can provide cost-effective strategies for the biotechnology industry to select promoters and other modular elements or a suppression vector, particularly when conducting in vivo experiments that use expensive animal models or other experiments that use primary cells that are difficult to isolate.

"According to a first embodiment, the present invention provides a plate for selection of a promoter sequence, wherein the plate comprises a first row of a plurality of loci and a second row of a plurality of loci, wherein each of the plurality of loci within the first row comprises a first lentiviral vector comprising: (a) a promoter sequence, e.g., a promoter sequence selected from the group consisting of sequences for human cytomegalovirus (hCMV), mouse cytomegalovirus (mCMV), human elongation factor 1 alpha (hEF1.alpha.), mouse elongation factor 1 alpha (mEF1.alpha.), CAG (a combination of the cytomegalovirus (CMV) early enhancer element and chicken beta-actin promoter), human phosphoglycerate kinase (hPGK), mouse phosphoglycerate kinase (mPGK), and human ubiquitin (UBC); and (b) optionally, a reporter sequence. In some embodiments, the lentiviral vector may further comprise one or more if not all of: a scaffolding sequence such as a miRNA scaffolding sequence, wherein the miRNA scaffolding sequence is derived from an endogenous pri-miRNA sequence; (d) a mature strand insert sequence; and (e) a star strand insert sequence. In some embodiments, the mature strand insert sequence and the star strand insert sequence are each 18-30 nucleotides in length, the mature strand insert sequence and the star strand insert sequence are each located within an miRNA scaffolding sequence and the mature strand insert sequence is at least 60% complementary to the star strand insert sequence. Further, in some embodiments, neither the mature strand insert sequence nor the star strand insert sequence comprise a sequence derived from an endogenous miRNA that comprises the pri-miRNA scaffolding; and each of the plurality of loci within the second row comprises a second lentiviral vector, wherein the second lentiviral vector is the same as the first lentiviral vector except that it comprises a different promoter sequence than in the first row, for example a different promoter sequence selected from the group consisting of sequences for hCMV, mCMV, hEF1.alpha., mEF1.alpha., CAG, hPGK, mPGK, and UBC.

"According to a second embodiment, the present invention provides a plate for selection of a promoter sequence, wherein the plate comprises a first row of a plurality of loci and a second row of a plurality of loci, wherein each of the plurality of loci within the first row comprises a first lentiviral vector comprising: (a) a promoter sequence, e.g., a promoter sequence selected from the group consisting of sequences for human cytomegalovirus (hCMV), mouse cytomegalovirus (mCMV), human elongation factor 1 alpha (hEF1.alpha.), mouse elongation factor 1 alpha (mEF1.alpha.), CAG (a combination of the cytomegalovirus (CMV) early enhancer element and chicken beta-actin promoter), mouse phosphoglycerate kinase (mPGK), and human ubiquitin (UBC); and (b) optionally, a reporter sequence. In some embodiments, the lentiviral vector may further comprise one or more if not all of: a scaffolding sequence such as a miRNA scaffolding sequence, wherein the miRNA scaffolding sequence is derived from an endogenous pri-miRNA sequence; (d) a mature strand insert sequence; and (e) a star strand insert sequence. In some embodiments, the mature strand insert sequence and the star strand insert sequence are each 18-30 nucleotides in length, the mature strand insert sequence and the star strand insert sequence are each located within an miRNA scaffolding sequence and the mature strand insert sequence is at least 60% complementary to the star strand insert sequence. Further, in some embodiments, neither the mature strand insert sequence nor the star strand insert sequence comprise a sequence derived from an endogenous miRNA that comprises the pri-miRNA scaffolding; and each of the plurality of loci within the second row comprises a second lentiviral vector, wherein the second lentiviral vector is the same as the first lentiviral vector except that it comprises a different promoter sequence than in the first row, for example a different promoter sequence selected from the group consisting of sequences for hCMV, mCMV, hEF1.alpha., mEF1.alpha., CAG, mPGK, and UBC.

"According to a third embodiment, the present invention provides a method for selecting and obtaining a suppression vector. For convenience and unless otherwise specified, the phrase 'suppression vector' may be used herein to refer to the vector that is designed to include a promoter that is selected through embodiments of the present invention. A suppression vector may be a lentiviral vector, but it may also be other types of expression vectors such as phages, plasmids or cosmids. In some embodiments, the expression vector comprises an RNA sequence such as ORF, shRNA or microRNA.

"Some methods of the present invention comprise: (a) transducing different cells from a cell line of interest with lentiviral vectors located at at least two different loci of a plate described herein; (b) measuring intensities of signals generated by activity in the cells, thereby establishing a plurality of measured intensities; selecting a promoter sequence for insertion into a suppression vector, thereby obtaining a selected promoter sequence, wherein the selected promoter sequence has the highest measured intensity at the lowest multiplicity of infection; and (d) obtaining a suppression vector. The method may also require that the highest measured intensity is greater than a predefined level, such as being measurable by a device or visible to the human eye. The suppression vector may comprise (i) the selected promoter sequence; (ii) optionally, a reporter sequence; (iii) an miRNA scaffolding sequence, wherein the miRNA scaffolding sequence is derived from an endogenous pri-miRNA sequence; (iv) a mature strand insert sequence; and (v) a star strand insert sequence, wherein the mature strand insert sequence and the star strand insert sequence are each 18-30 or 18-23 or 19-23 nucleotides in length, the mature strand insert sequence and the star strand insert sequence are each located within the miRNA scaffolding sequence and the mature strand insert sequence is at least 60% complementary to the star strand insert sequence, and neither the mature strand insert sequence nor the star strand insert sequence comprise a sequence derived from the endogenous pri-miRNA.

"As noted above, the suppression vector, may for example, comprise the promoter that is selected in step . It also may comprise a scaffolding sequence that is the same as a scaffolding sequence within the lentiviral vectors of step (a). Alternatively, the suppression vector may contain a different scaffolding or no scaffolding. Moreover, in some embodiments, the lentiviral vector contains a non-targeting control sequence, but the suppression vector comprises a sequence designed to target or silence a nucleotide sequence within a cell or organism. The suppression vectors may then be used to introduce genetic material into cells in vitro or into an organism in vivo.

"According to a fourth embodiment, the present invention provides a lentiviral vector comprising: (a) a promoter sequence selected from the group consisting of sequences for hCMV, mCMV, hEF1.alpha., mEF1.alpha., CAG, hPGK, mPGK, and UBC; and (b) a reporter sequence. The lentiviral vector may further comprise one or more of a scaffolding sequence, a mature strand insert sequence and a star strand insert sequence. Optionally, within the lentiviral vector, the promoter sequence may be selected from the group consisting of sequences for hCMV, mCMV, hEF1.alpha., mEF1.alpha., CAG, mPGK, and UBC

"According to a fifth embodiment, the present invention provides a lentiviral vector comprising: (a) a promoter sequence selected from the group consisting of sequences for hCMV, mCMV, hEF1.alpha., mEF1.alpha., CAG, hPGK, mPGK, and UBC; (b) optionally, a reporter sequence; an miRNA scaffolding sequence, wherein the miRNA scaffolding sequence is derived from an endogenous pri-miRNA sequence; (d) a mature strand insert sequence; and (e) a star strand insert sequence, wherein the mature strand insert sequence and the star strand insert sequence are each 18-23 nucleotides in length, and the mature strand insert sequence and the star strand insert sequence are each located within the miRNA scaffolding and the mature strand insert sequence is at least 60% complementary to the star strand insert sequence, and neither the mature strand insert sequence nor the star strand insert sequence comprise a sequence derived from the endogenous pri-miRNA. Optionally, within the lentiviral vector, the promoter sequence may be selected from the group consisting of sequences for hCMV, mCMV, hEF1.alpha., mEF1.alpha., CAG, mPGK, and UBC.

"According to a sixth embodiment, the present invention provides a kit for suppressing gene expression comprising a plurality of lentiviral vectors described herein, wherein the plurality of lentiviral vectors comprises at least two lentiviral vectors that have different promoter sequences. Preferably, although having different promoter sequences they otherwise have the same sequences.

"According to a seventh embodiment, the present invention provides a plate for selection of a promoter sequence, wherein the plate comprises a first row of a plurality of loci and a second row of a plurality of loci, wherein each of at least two of the plurality of loci within the first row comprises a first lentiviral vector comprising: (i) a first promoter sequence; and (ii) a reporter sequence; and at least two of the plurality of loci within the second row each comprises a second lentiviral vector, wherein the second lentiviral vector is the same as the first lentiviral vector except that it does not comprise the first promoter sequence and comprises a second promoter sequence that is different from the first promoter sequence.

"The plates and various other embodiments of the present invention may be advantageous for use by researchers who do not necessarily have a priori knowledge of optimal promoters for their biological cells or animal models of interest because they offer cost-effective means by which to design lentiviral particles. Thus, through the use of one or more of the embodiments of the present invention, one may achieve one or more of the following advantages: increased efficiency when testing promoters; increased efficiency when choosing reporters; increased efficiency of transduction; and an improved ability to develop and to use customized vectors that have a likelihood of success.

BRIEF DESCRIPTION OF THE FIGURES

"FIG. 1 is a representation of promoter activity as measured by GFP fluorescence from the results of example 1 in PC3 cells.

"FIG. 2 is a representation of promoter activity as measured by GFP fluorescence from the results of example 1 in OVCAR8 cells.

"FIG. 3 is a representation of promoter activity as measured by GFP fluorescence from the results of example 2 in A549 cells.

"FIG. 4 is a representation of promoter activity as measured by GFP fluorescence from the results of example 2 in Jurkat cells.

"FIG. 5 is a representation of promoter activity as measured by GFP fluorescence from the results of example 3 in NIH3T3 cells and a measure of relative GAPDH expression following expression from four different promoters of an shRNA targeting GAPDH.

"FIG. 6 is a representation of promoter activity as measured by GFP fluorescence from the results of example 3 in A549 cells and a measure of relative GAPDH expression following expression from four different promoters of an shRNA targeting GAPDH.

"FIG. 7 is a representation of serial dilutions of test vectors in a plate according to an embodiment of the present invention.

"FIG. 8 is a representation of the GFP fluorescence intensities from the results in example 5 in A549 cells, HEK293T cells, and Jurkat cells following transduction with serial dilutions of test vectors in a plate according to an embodiment of the present invention.

"FIG. 9 is a representation of the GFP fluorescence intensities in A549 cells following transduction of test vectors in a plate according to an embodiment of the present invention.

"FIG. 10 is a representation of a quantitative analysis of gene silencing with three shRNAs targeting the RHOA gene and expressed by the mouse CMV and human CMV promoters in A549 cells.

"FIG. 11 is a representation of a quantitative analysis of gene silencing with three shRNAs targeting the RHOA gene and expressed by the mouse EF1.alpha. and human CMV promoters in Jurkat cells.

"FIG. 12 is a representation of an example of configuration options for vectors of the present invention."

For more information, see this patent application: Wakefield, John. Optimization of Vectors for Effective Delivery and Expression of Genetic Content. Filed October 2, 2012 and posted August 7, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=2034&p=41&f=G&l=50&d=PG01&S1=20140731.PD.&OS=PD/20140731&RS=PD/20140731

Keywords for this news article include: Biotechnology, Viral, Kinase, Genetics, Virology, Immunology, Leukocytes, DNA Viruses, Gene Therapy, Jurkat Cells, Immune System, T-Lymphocytes, Bioengineering, Betaherpesvirinae, DNA Virus Infections, Enzymes and Coenzymes, Herpesviridae Infections, Cytomegalovirus Infections, Thermo Fisher Scientific Biosciences Inc..

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Source: Gene Therapy Weekly


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