Mammalian ncRNA Expression Self-Inactivating MMLV Vector

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.

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.

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 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.

CMV promoter: Human cytomegalovirus immediate early promoter. It drives transcription of viral RNA in packaging cells. This RNA is then packaged into live virus.

MMLV 5' LTR-ΔU3: A deleted version of the MMLV retrovirus 5' long terminal repeat. In wildtype MMLV retrovirus, 5' LTR and 3' LTR are essentially identical in sequence. They reside on two ends of the viral genome and point in the same direction. Upon viral integration, the 3' LTR sequence is copied onto the 5' LTR. The LTRs carry both promoter and polyadenylation function, such that the 5' LTR acts as a promoter to drive the transcription of the viral genome, while the 3' LTR acts as a polyadenylation signal to terminate the upstream transcript. On our vector, MMLV 5' LTR-ΔU3 is deleted for a region that is required for the LTR's promoter activity. This does not affect the production of viral RNA during packaging because the promoter function is supplemented by the CMV promoter engineered upstream of Δ5' LTR.

Ψ plus pack2: MMLV retrovirus packaging signal required for the packaging of viral RNA into virus.

Promoter: The promoter driving expression of your non-coding RNA is placed here.

Non-coding RNA: The non-coding RNA of your interest is placed here.

WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element. It enhances viral RNA stability in packaging cells, leading to higher titer of packaged virus.

MMLV 3' LTR-ΔU3: A truncated version of the MMLV retrovirus 3' long terminal repeat. This leads to the self-inactivation of the promoter activity of the 5' LTR upon viral vector integration into the host genome (due to the fact that 3' LTR is copied onto 5' LTR during viral integration). The polyadenylation signal contained in MMLV 3' LTR-ΔU3 serves to terminate all upstream transcripts produced both during viral packaging and after viral integration into the host genome.

SV40 late pA: Simian virus 40 late polyadenylation signal. It further facilitates transcriptional termination after the 3' LTR during packaging. This elevates the level of functional viral RNA in packaging cells, thus improving viral titer.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

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.

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.

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.

CMV promoter: Human cytomegalovirus immediate early promoter. It drives transcription of viral RNA in packaging cells. This RNA is then packaged into live virus.

MMLV 5' LTR-ΔU3: A deleted version of the MMLV retrovirus 5' long terminal repeat. In wildtype MMLV retrovirus, 5' LTR and 3' LTR are essentially identical in sequence. They reside on two ends of the viral genome and point in the same direction. Upon viral integration, the 3' LTR sequence is copied onto the 5' LTR. The LTRs carry both promoter and polyadenylation function, such that the 5' LTR acts as a promoter to drive the transcription of the viral genome, while the 3' LTR acts as a polyadenylation signal to terminate the upstream transcript. On our vector, MMLV 5' LTR-ΔU3 is deleted for a region that is required for the LTR's promoter activity. This does not affect the production of viral RNA during packaging because the promoter function is supplemented by the CMV promoter engineered upstream of Δ5' LTR.

Ψ plus pack2: MMLV retrovirus packaging signal required for the packaging of viral RNA into virus.

Promoter: The promoter driving expression of your non-coding RNA is placed here.

Non-coding RNA: The non-coding RNA of your interest is placed here.

WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element. It enhances viral RNA stability in packaging cells, leading to higher titer of packaged virus.

MMLV 3' LTR-ΔU3: A truncated version of the MMLV retrovirus 3' long terminal repeat. This leads to the self-inactivation of the promoter activity of the 5' LTR upon viral vector integration into the host genome (due to the fact that 3' LTR is copied onto 5' LTR during viral integration). The polyadenylation signal contained in MMLV 3' LTR-ΔU3 serves to terminate all upstream transcripts produced both during viral packaging and after viral integration into the host genome.

SV40 late pA: Simian virus 40 late polyadenylation signal. It further facilitates transcriptional termination after the 3' LTR during packaging. This elevates the level of functional viral RNA in packaging cells, thus improving viral titer.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.