Mammalian ncRNA Expression Self-Inactivating MMLV Vector

Overview

The MMLV retroviral vector system is an efficient vehicle for introducing non-coding RNAs permanently into mammalian cells. Non-coding RNAs include a wide variety of short (<30 nucleotides) and long (>200 nucleotides) functional RNA molecules such as micro RNAs (miRNAs), small interfering RNAs (siRNAs), piwi-interacting RNAs (piRNAs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), large intergenic non-coding RNAs (lincRNAs), intronic long non-coding RNAs (intronic lncRNAs), natural antisense transcripts (NATs), enhancer RNAs (eRNAs) and promoter-associated RNAs (PARs), none of which are translated into proteins, however have been found to play important roles in many cellular processes such as DNA replication, epigenetic regulation, transcriptional and post-transcriptional regulation and translation regulation.

MMLV, a retroviral vector derived from Moloney murine leukemia virus, is a plus-strand linear RNA virus that exhibits efficient genomic integration. While our wildtype MMLV retrovirus expression vector utilizes the ubiquitous promoter function in the 5' long terminal repeat (LTR) of wildtype MMLV genome for driving expression of the non-coding RNA, the self-inactivating MMLV retrovirus expression vector allows users to select any promoter of their choice for driving non-coding RNA expression. This is achieved by the deletion of the U3 region in the MMLV 3’ LTR which self-inactivates the promoter activity in the 5' LTR by a copying mechanism during viral genome integration. This not only provides users with the flexibility to add their promoter of choice for driving non-coding RNA expression but also eliminates the risk of oncogenic activation of adjacent genes upon vector integration, thereby enabling such vectors to have a higher safety profile compared to wildtype MMLV vectors.

A self-inactivating MMLV retroviral vector is first constructed as a plasmid in E. coli. It is then transfected into packaging cells along with several helper plasmids. Inside the packaging cells, vector DNA located between the LTRs is transcribed into RNA, and viral proteins expressed by the helper plasmids further package the RNA into virus. Live virus is then released into the supernatant, which can be used to infect target cells directly or after concentration.

When the virus is added to target cells, the RNA cargo is shuttled into cells where it is reverse transcribed into DNA and randomly integrated in the host genome. The non-coding RNA sequence that was placed in-between the two LTRs during vector construction is permanently inserted into host DNA alongside the rest of viral genome.

By design, self-inactivating MMLV retroviral vectors lack the genes required for viral packaging and transduction (these genes are carried by helper plasmids or integrated into packaging cells instead). As a result, viruses produced from these vectors have the important safety feature of being replication incompetent (meaning that they can transduce target cells but cannot replicate in them).

For further information about this vector system, please refer to the papers below.

References Topic
Cell. 157:77 (2014) Review on non-coding RNAs
Front Genet. 6:2 (2015) Review on functionality of non-coding RNAs
PLoS One. 8:e77070 (2013) Retrovirus-mediated expression of long non-coding RNA
J Virol. 61:1639 (1987) Extended packaging signal increases the titer of MMLV vectors
Gene Ther. 7:1063 (2000) Tropism of MMLV vectors depends on packaging cell lines
Proc Natl Acad Sci USA. 83:3194 (1986) Designing of self-inactivating retroviral vectors for gene transfer into mammalian cells

Highlights

The SIN MMLV retrovirus non-coding RNA expression vector is optimized for high copy number replication in E. coli, high-titer packaging of live virus, efficient viral transduction of a wide range of cells, efficient vector integration into the host genome, and high-level expression.

Advantages

Permanent integration of vector DNA: Conventional transfection results in almost entirely transient delivery of DNA into host cells due to the loss of DNA over time. This problem is especially prominent in rapidly dividing cells. In contrast, retroviral transduction can deliver non-coding RNAs permanently into host cells due to integration of the viral vector into the host genome.

Broad tropism: Our packaging system adds the VSV-G envelop protein to the viral surface. This protein has broad tropism. As a result, cells from all commonly used mammalian species such as human, mouse and rat can be transduced. Furthermore, many specific cell types can be transduced, though our vector has difficulty transducing non-dividing cells (see disadvantages below).

Customizable internal promoter: Our vector is designed to self-inactivate the promoter activity in its 5' LTR upon integration into the genome. As a result, users can put in their own promoter to drive their non-coding RNA within the vector. This is a distinct advantage over our wildtype MMLV retrovirus vectors, which rely on the promoter function of 5' LTR to drive the ubiquitous expression.

Relative uniformity of delivery: Generally, viral transduction can deliver vectors into cells in a relatively uniform manner. In contrast, conventional transfection of plasmid vectors can be highly non-uniform, with some cells receiving a lot of copies while other cells receiving few copies or none.

Effectiveness in vitro and in vivo: While our vector is mostly used for in vitro transduction of cultured cells, it can also be used to transduce cells in live animals.

Safety: The safety of our vector is ensured by two features. One is the partitioning of genes required for viral packaging and transduction into several helper plasmids; the other is self-inactivation of the promoter activity in the 5' LTR upon vector integration. As a result, it is essentially impossible for replication competent virus to emerge during packaging and transduction. The health risk of working with our vector is therefore minimal.

Disadvantages

Moderate viral titer: Viral titer from our vector reaches ~107 TU/ml in the supernatant of packaging cells without further concentration. This is about an order of magnitude lower than our lentiviral vectors.

More limited cargo space than wildtype MMLV: The MMLV retroviral genome is ~8.3 kb. In our vector, the components necessary for viral packaging and transduction occupy ~2.6-3 kb, which leaves only ~5.3-5.7 kb to accommodate the user's DNA of interest, including both the CAR expression cassette and the promoter.

Difficulty transducing non-dividing cells: Our vector has difficulty transducing non-dividing cells.

Technical complexity: The use of MMLV retroviral vectors requires the production of live virus in packaging cells followed by the measurement of viral titer. These procedures are technically demanding and time consuming relative to conventional plasmid transfection.

Mammalian ncRNA Expression Self-Inactivating MMLV Vector

Overview

The MMLV retroviral vector system is an efficient vehicle for introducing non-coding RNAs permanently into mammalian cells. Non-coding RNAs include a wide variety of short (<30 nucleotides) and long (>200 nucleotides) functional RNA molecules such as micro RNAs (miRNAs), small interfering RNAs (siRNAs), piwi-interacting RNAs (piRNAs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), large intergenic non-coding RNAs (lincRNAs), intronic long non-coding RNAs (intronic lncRNAs), natural antisense transcripts (NATs), enhancer RNAs (eRNAs) and promoter-associated RNAs (PARs), none of which are translated into proteins, however have been found to play important roles in many cellular processes such as DNA replication, epigenetic regulation, transcriptional and post-transcriptional regulation and translation regulation.

MMLV, a retroviral vector derived from Moloney murine leukemia virus, is a plus-strand linear RNA virus that exhibits efficient genomic integration. While our wildtype MMLV retrovirus expression vector utilizes the ubiquitous promoter function in the 5' long terminal repeat (LTR) of wildtype MMLV genome for driving expression of the non-coding RNA, the self-inactivating MMLV retrovirus expression vector allows users to select any promoter of their choice for driving non-coding RNA expression. This is achieved by the deletion of the U3 region in the MMLV 3’ LTR which self-inactivates the promoter activity in the 5' LTR by a copying mechanism during viral genome integration. This not only provides users with the flexibility to add their promoter of choice for driving non-coding RNA expression but also eliminates the risk of oncogenic activation of adjacent genes upon vector integration, thereby enabling such vectors to have a higher safety profile compared to wildtype MMLV vectors.

A self-inactivating MMLV retroviral vector is first constructed as a plasmid in E. coli. It is then transfected into packaging cells along with several helper plasmids. Inside the packaging cells, vector DNA located between the LTRs is transcribed into RNA, and viral proteins expressed by the helper plasmids further package the RNA into virus. Live virus is then released into the supernatant, which can be used to infect target cells directly or after concentration.

When the virus is added to target cells, the RNA cargo is shuttled into cells where it is reverse transcribed into DNA and randomly integrated in the host genome. The non-coding RNA sequence that was placed in-between the two LTRs during vector construction is permanently inserted into host DNA alongside the rest of viral genome.

By design, self-inactivating MMLV retroviral vectors lack the genes required for viral packaging and transduction (these genes are carried by helper plasmids or integrated into packaging cells instead). As a result, viruses produced from these vectors have the important safety feature of being replication incompetent (meaning that they can transduce target cells but cannot replicate in them).

For further information about this vector system, please refer to the papers below.

References Topic
Cell. 157:77 (2014) Review on non-coding RNAs
Front Genet. 6:2 (2015) Review on functionality of non-coding RNAs
PLoS One. 8:e77070 (2013) Retrovirus-mediated expression of long non-coding RNA
J Virol. 61:1639 (1987) Extended packaging signal increases the titer of MMLV vectors
Gene Ther. 7:1063 (2000) Tropism of MMLV vectors depends on packaging cell lines
Proc Natl Acad Sci USA. 83:3194 (1986) Designing of self-inactivating retroviral vectors for gene transfer into mammalian cells

Highlights

The SIN MMLV retrovirus non-coding RNA expression vector is optimized for high copy number replication in E. coli, high-titer packaging of live virus, efficient viral transduction of a wide range of cells, efficient vector integration into the host genome, and high-level expression.

Advantages

Permanent integration of vector DNA: Conventional transfection results in almost entirely transient delivery of DNA into host cells due to the loss of DNA over time. This problem is especially prominent in rapidly dividing cells. In contrast, retroviral transduction can deliver non-coding RNAs permanently into host cells due to integration of the viral vector into the host genome.

Broad tropism: Our packaging system adds the VSV-G envelop protein to the viral surface. This protein has broad tropism. As a result, cells from all commonly used mammalian species such as human, mouse and rat can be transduced. Furthermore, many specific cell types can be transduced, though our vector has difficulty transducing non-dividing cells (see disadvantages below).

Customizable internal promoter: Our vector is designed to self-inactivate the promoter activity in its 5' LTR upon integration into the genome. As a result, users can put in their own promoter to drive their non-coding RNA within the vector. This is a distinct advantage over our wildtype MMLV retrovirus vectors, which rely on the promoter function of 5' LTR to drive the ubiquitous expression.

Relative uniformity of delivery: Generally, viral transduction can deliver vectors into cells in a relatively uniform manner. In contrast, conventional transfection of plasmid vectors can be highly non-uniform, with some cells receiving a lot of copies while other cells receiving few copies or none.

Effectiveness in vitro and in vivo: While our vector is mostly used for in vitro transduction of cultured cells, it can also be used to transduce cells in live animals.

Safety: The safety of our vector is ensured by two features. One is the partitioning of genes required for viral packaging and transduction into several helper plasmids; the other is self-inactivation of the promoter activity in the 5' LTR upon vector integration. As a result, it is essentially impossible for replication competent virus to emerge during packaging and transduction. The health risk of working with our vector is therefore minimal.

Disadvantages

Moderate viral titer: Viral titer from our vector reaches ~107 TU/ml in the supernatant of packaging cells without further concentration. This is about an order of magnitude lower than our lentiviral vectors.

More limited cargo space than wildtype MMLV: The wildtype MMLV retroviral genome is ~8.3 kb. In our vector, the components necessary for viral packaging and transduction occupy ~2.6-3 kb, which leaves only ~5.3-5.7 kb to accommodate the user's DNA of interest, including both the non-coding RNA and the promoter.

Difficulty transducing non-dividing cells: Our vector has difficulty transducing non-dividing cells.

Technical complexity: The use of MMLV retroviral vectors requires the production of live virus in packaging cells followed by the measurement of viral titer. These procedures are technically demanding and time consuming relative to conventional plasmid transfection.

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