Patent ID: 12215339

DETAILED DESCRIPTION

The present subject matter relates to enhanced production of lentiviral particles which may be cultured, harvested and utilized to introduce polynucleotide sequences of interest into target cells. A lentiviral particle, also referred to as a lentiviral vector or simply “lentivirus”, is an enveloped viral package contains an expressible polynucleotide sequence, and which is capable of penetrating a target cell, thereby carrying the expressible sequence into the cell. Preferably, the package is enveloped with a viral envelope protein from another viral species or, including non-lentiviruses, or an engineered envelope protein, which changes the infectivity of the native lentivirus. The harvested lentivirus may be used in a wide variety of applications, for example vaccine production, gene therapy, polypeptide delivery, siRNA delivery, as well as delivery of ribozymes, anti-sense, and other functional polynucleotides.

The present inventive subject matter provides a novel lentivirus packaging system that results in enhanced production of lentivirus upon transfection of a transfer plasmid with the packaging system into a suitable host cell. A vector comprising the TRE promotor and tTA was constructed and evaluated as a positive feedback loop. Lentivirus packaging systems comprising a synthetic gene network with the positive feedback loop were engineered. Surprisingly, the it was found that co-transfection of the novel plasmid packaging system engineered with a PFL and a transfer plasmid into a host cell resulted in enhanced Lentivirus production with viral titers dramatically increased over production of Lentivirus utilizing a conventional third generation packaging system.

According to one embodiment, methods for enhancing production of lentivirus n a host cell are disclosed. The methods comprise providing a transfer plasmid comprising a nucleic acid of interest, for example a target gene or a gene modulator; providing a helper plasmid comprising tetracycline transactivator (tTA) under the control of tetracycline responsive element (TRE) (TRE-tTA) as the positive feedback loop, and co-transfecting the host cell with (TG-TRE) and the transfer plasmid. In more specific embodiments, the method further comprises co-transfecting the host cell with a CMV promotor controlled tTA (CMV-tTA) helper plasmid and an envelope plasmid. The envelope plasmid may be any envelope plasmid or combination of envelope plasmids. According to a very specific embodiment, the envelope plasmid comprises CMV-VSV-G. According to other specific embodiments, the methods further comprise further comprise co-transfecting the host cell with TRE-ΔR8.2-Δvpr plasmid. To reduce the possibility of viral recombination, the packaging vectors may be further divided to provide a five-helper plasmid system. In very specific embodiments the TRE-ΔR8.2-Δvpr plasmid is replaced with a TRE-ΔR8.2-Δvpr,rev,nef helper plasmid and a TRE-rev helper plasmid.

The host cell may be any cell suitable for transfection by plasmids and recombination of lentivirus. According to specific embodiments, the host cell comprises human embryonic kidney cells 293 (HEK293). Although generally mammalian cells, and in particular 293T cells are most common for purposes of producing and harvesting lentivirus, a person of ordinary skill in the art will understand the requisites to selection of a suitable host cell.

Generally in order to produce recombined lentivirus, the host cell is transfected with a transfer plasmid, one or more packaging plasmids, and an envelope plasmid. One embodiment is directed to a novel lentivirus packaging system comprising the PFL plasmid (TRE-tTA) as one of the packaging plasmids. In other embodiments the system further comprises a plasmid comprising (CMV-tTA), and at least one envelope vector, for example the CMV-VSV-G plasmid. Any envelope plasmid, however, is suitable and selection may be based on a desired tropism with a cell/tissue relevant to a therapeutic end use. In more specific embodiments the system further comprises at least one plasmid comprising a TRE promotor controlling a subset of HIV-1 genes. This is the basis for the positive feedback loop. In very specific embodiments the subset of HIV-1 genes excludes LTRs, vpr and env. In more specific embodiments the TRE promotor controlling a subset of HIV-1 genes comprises a TRE-ΔR8.2-Δvpr plasmid. In other specific embodiments suitable for safer therapeutic uses, the TRE-ΔR8.2-Δvpr plasmid is replaced with two plasmids: a TRE-ΔR8.2-Δvpr,rev,nef plasmid and a TRE-rev plasmid. Methods for enhanced recombinant production of lentivirus in a host cell are provided by co-transfecting a suitable host cell with the system comprising at least one envelope vector, a transfer plasmid, a PFL TRE-tTA plasmid, and a TRE-ΔR8.2-Δvpr plasmid. In other specific methods enhanced recombinant production of lentivirus is provided by co-transfecting a suitable host cell with the system comprising at least one envelope vector, a transfer plasmid, the PFL TRE-tTA plasmid, at least one transfer plasmid, a TRE-ΔR8.2-Δvpr,rev,nef plasmid, and a TRE-rev plasmid.

A transfer plasmid may be any known transfer plasmid and a person of ordinary skill in the art will understand that the specific identity of the transfer plasmid is determined by desired end use. For exemplary purposes and to demonstrate the enhanced viral production, specific transfer plasmids selected from from pCSII-mVenus-hGeminin, ExEGFP-Lv105, and pSMPUW-CMV-mRFP were co-transfected with an embodiment of the inventive packaging system. For purposes of therapeutic applications, compositions of lentivirus produced from recombination of the transfer plasmid and at least one envelope plasmid with embodiments of the plasmid packaging system may be formulated by harvesting lentivirus from the host cell and optionally concentrating it via known methods such as centrifugation.

Methods for treating a patient suffering from a disorder characterized by deficiency in expression of a target gene (TG) in a cell are also provided.

REFERENCES

Adfenoleukodystrophy: Cartier, N. et al.Science326, 818-823 (2009).beta-thalassaemia: Cavazzana-Calvo, M. et al.Nature467, 318-322 (2010).

Gene replacement therapy (GRT) may utilize lentivirus to transduce a target cell to deliver a polynucleotide of interest. Zhao et al. Gene Therapy 12:311-319, 2005, the entire disclosure of which is incorporated herein by this reference, discloses GRT effectuated by HIV-1 based lentivirus. Specific examples of diseases characterized by deficiencies in genes that may be treated by HIV-based lentivirus include Adrenoleukodystrophy (Cartier, N. et al. Science 326, 818-823 (2009), incorporated herein by reference) and β-thalassaemia (Cavazzana-Calvo, M. et al. Nature 467, 318-322, incorporated herein by reference). Embodiments provide methods for treating diseases and disorders characterized by a functional gene expression product deficiency that may be treated by providing target nucleic acid, e.g. a target gene into the genome of the patient. Lentivirus is advantageous as a delivery vector in GRT because it can transduce both replicating and non-replicating cells. Methods comprise co-transfecting the cell with a lentivirus transfer plasmid comprising, for example, a TG or a modulator of the TG, and a set of packaging plasmids comprising a TRE-tTA plasmid, a CMV-tTA plasmid, at least one envelope plasmid, and at least one plasmid comprising a TRE promotor controlling a subset of HIV-1 genes. The inventive attribute resulting from practice of embodiments of the instant invention that is generally applicable to therapeutic utility is the dramatic increase in viral titer resulting from engineering of the positive feedback loop into the plasmid packaging system.

EXAMPLES

The following Examples are provided solely to illustrate particular embodiments and aspects of the invention, and should not be construed as limiting the scope thereof, as defined by the appended claims. The “materials” disclosure set forth below applies to Examples 2-6.

Materials

pCSII-SYN-Venus, pCSII-Sox2, pCMV-VSV-G-RSV-rev plasmids were obtained from A Miyoshi (Keio University). pCSII-mVenus-hGeminin and pCSII-mCherry-hCDT plasmids were obtained from A. Miyawaki (RIKEN BSI). pcDNA3-mRFP plasmid was obtained from D. Golenbock (University of Massachusetts Medical School, Addgene plasmid #13032). tet operator plasmid was obtained from L. Chin (University of Texas, Addgene plasmid #8901). FUW-OSKM and TetO-FUW-OSKM were obtained from R. Jaenisch (Whitehead Institute, Addgene plasmid #20328, #20321). pSMPUW, pCgpV, and pRSV-rev were obtained from CellBioLabs. ExEGFP-LV100 plasmid was obtained from GeneCopoeia. Anti-Oct4 antibody was obtained fromCellSignaling Technology.

Example 1

Plasmid Construction.

TRE-ΔR8.2-Δvpr was constructed with the deletion of vpr gene from pPTK (see Kafri, T. et al.J. Vir.73, 576-584 (1999), incorporated herein by this reference) by PCR. The PCR fragment was phospholilated by ploynucleotide kinase (Ambion) and ligated. The following primers were used:

5′-AAAGGTGGCTTTATCTGTTTTGGTTTTATTAATG-3′and5′-TAAGAGTGAAGCTGTTAGACATTTTCC-3′
(see An, D. S.J. Vir.73, 7671-7677 (1999), incorporated herein by this reference). The genes of rev, nef were further deleted by digestion with SalI and BamHI from TRE-ΔR8.2-Δvpr to construct TRE-ΔR8.2-Δvpr,rev,nef. The digested fragment was blunted and then ligated. For the construction of TRE-tTA, tTA was digested from pTet-Off (Clontech) and inserted in EcoRI and HindIII sites of tet operator plasmid (Chin et al.Nature400, 468-472 (1999), incorporated herein by this reference). To construct TRE-rev, rev was digested from pRSV-rev (CellBiolab) and inserted in EcoRI and HindIII sites of pTRE-tight (Clontech). CMV promoter was digested with BglII and BamHI from pcDNA3.1 (Life Technologies) and inserted in BamHI site of pSMPUW (CellBiolab), and then mRFP was amplified from pcDNA3-mRFP by PCR and further inserted in BamHI and SalI sites to construct pSMPUW-CMV-mRFP. The following primers were used:

5′-ACGAGATCTACCATGGCCTCCTCCGA-3′and5′-ACGCGTCGACTTAGGCGCCGGTGGA-3′.
For the construction of pTRE-SMPUW-CMV-mRFP, TRE-tight promoter was amplified from pTRE-tight (Clontech) by PCR and the fragment was inserted in speI site of pSMPUW-CMV-mRFP. The following primers were used:

5′-GCACTAGTTTACTCCCTATCAGTGAT-3′and5′-CGACTAGTCTCCAGGCGATCTGACG-3′.
(See Campbell et al.Proc. Natl. Acad. Sci. USA99, 7877-7882 (2002), incorporated herein by this reference.)

Example 2

Lentiviral Production and Titration

Lentiviruses were generated by transient co-transfection of a transfer plasmid (2.4 μg) with CMV-VSV-G (0.8 μg), CMV-tTA 0.8 μg), TRE-ΔR8.2-Δvpr (0.8 μg), and TRE-tTA (1.6 μg) into HEK293Ta cells (GeneCopoeia) for the four plasmid PFL system. For the five plasmids PFL system, TRE-rev (0.8 μg) and TRE-ΔR8.2-Δvpr,rev,nef (0.8 μg) were used instead of TRE-ΔR8.2-Δvpr. For the conventional third generation system, pCgpV (0.8 μg) and pCMV-VS V-G-RSV-rev 0.8 μg) were used as packaging plasmids. The medium was changed with DMEM containing 1 mM sodium butyrate and 4 mM caffeine after 6-12 hr transfection to stimulate lentivirus production (2011 Elis, HumGeneTherapy). The lentiviruses were concentrated to 100 times by Lenti-X Concentrator (Clontech). For injection into mouse brain, lentiviruses were concentrated to 1000 times by ultracentrifugation (Miyoshi et al.J. Vir.72, 8150-8157 (1998), incorporated herein by this reference). The titers of viruses were determined by infection of HEK293Ta cells, seeded in 96-well plates at 104cells/well with serial dilutions of the vector stock. The numbers of EGFP- or mRFP-positive cells were counted to calculate the titer. qRT-PCR was run for the quantification of titers of the lentiviruses made by transfer plasmids of pCSII-SYN-Venus, pCSII-Sox2, pCSII-mVenus-hGeminin, or pCSII-mCherry-hCDT. RNAs were purified from concentrated lentiviruses with TRT REAGENT RT (Molecular Research Center, Inc.) and treated with RQ1-DNase (Promega) to remove contaminated plasmid DNAs. Reverse transcription reactions were done with GoScript system (Promega). qRT-PCR reaction was done with Fast SYBR Green Master Mix (Life Technologies) by StepOnePlus real time PCR system (Life Technologies). The obtained data was analyzed with the comparative CT method, and the values of 2dCTof non-RT samples were subtracted from the values of 2dCTof RT samples to eliminate the effect of contamination of the plasmid DNA. The titers were determined by the comparison of the 2dCTvalues to that from lentiviruses made by the transfer plasmid of ExEGFP-LV100. The following primers were used for the amplification of EGFP:

5′-ACGAGATCTACCATGGCCTCCTCCGA-3′and5′-ACGCGTCGACTTAGGCGCCGGTGGA-3′.
The following primers were used for the amplification of WPRE:

5′-ACGAGATCTACCATGGCCTCCTCCGA-3′and5′-ACGCGTCGACTTAGGCGCCGGTGGA-3′.

Example 3

Construction and Evaluation of the Positive Feedback Loop

A plasmid with tetracycline transactivator (tTA) under the control of tetracycline responsive element (TRE) was first constructed. tTA can hind to the TRE promoter and activate its own transcription without addition of tetracycline or doxycycline. Therefore, the plasmid creates a synthetic gene network with PFL to amplify the expression of tTA and any other target genes under the control of TRE promoter (FIG.3A). This PFL was first tested with luciferase activity. The TRE promoter was connected to luciferase gene (TRE-luc), and the luciferase assay was done by transient co-transfection of TRE-luc with the CMV promoter controlled tTA containing plasmid (CMV-tTA) and/or the TRE promoter controlled tTA containing plasmid (TRE-tTA) in HEK293Ta cells (FIG.3B). The average luminescence of luciferase expressing cells co-transfected with CMV-rTA, TRE-tTA, or CMV-tTA and TRE-tTA were 85.1±7.0, 1823.6±118.7, and 4135.6±81.7, respectively (A.U.; n=3) (FIG.3C). Co-transfection of TRE-luc with CMV-tTA and TRE-tTA gave 50 times more luciferase activity than co-transfection of TRE-luc and CMV-tTA. The TRE promoter activity with the PFL was further compared to human ubiquitin promoter (hUbC) (see Carey, B. W. et al.Proc. Natl. Acad. Sci. USA106, 157-162 (2009), incorporated herein by this reference) and showed extensively enhanced signal over hUbC (FIG.3BandFIG.3D).

Example 4

Development of a Lentivirus Packaging System with the Positive Feedback Loop

Based on the PFL of tTA and TRE promoter, a new lentivirus packaging system was developed.FIG.1Ais a schematic diagram of the gene network of the helper plasmids (gray arrows inFIG.1A). The packaging system containing four helper plasmids that incorporates: (i) TRE-ΔR8.2-Δvpr (pPTK-Δvpr) contains TRE promoter which controls all HIV-1 genes except LTRs, vpr, and env (Kafri, T. et al.J. Vir.73, 576-584 (1999) and Stewart, S. A. et al.RNA9, 493-501 (2003), the entire disclosures of which are incorporated herein by this reference); (ii) CMV-VSV-G (pCMV-VSV-G) is the envelope vector; (iii) CMV-tTA (pTetOff) is for the expression of the tTA transactivator under the control of CMV promoter; and (iv) TRE-tTA (pBS-TRE) contains tTA under the control of TRE promoter and establishes a PFL. Using these packaging plasmids, titers of lentiviruses made by the packaging system with and without TRE-tTA were compared. The packaging system without TRE-tTA was designated as a non-PFL (NPFL) system and the packaging system with TRE-tTA was designated as a PFL system. Compared to the lentiviruses made by the NPFL system, the lentivirus made by the PFL system had more than 3,000 times higher titer (FIG.1B). In the NPFL system, dose increase of the quantity of CMV-tTA plasmid resulted in a two-fold augmentation of the titer (FIG.1C). In comparison, the dose-dependent increase of the titers was more drastic in the PFL system. The gradual increase of the amount of TRE-tTA resulted in an abrupt increase of virus production when the amount exceeded the threshold (FIG.1D).

Example 5

Evaluation of the Inventive Packaging System Using Three Specific Transfer Plasmids

The novel packaging system was further evaluated with three different transfer plasmids. Lentivirus packaging was done by co-transfection of each transfer plasmid with the NPFL system, the PFL system, or conventional third generation packaging system in HEK293Ta host cells. The effect of the PFL was quite potent (FIG.2A-C). The titers of lentiviruses made with the PFL system were 250-, 90,000-, and 2,400-fold higher compared to the titers of lentiviruses made with the NPFL system for pCSII-mVenus-hGeminin, ExEGFP-Lv105, and pSMPUW-CMV-mRFP transfer plasmids, respectively (FIG.2A-C). The titers of the lentiviruses made with the PFL system were also significantly higher than that of the lentiviruses made with conventional third generation packaging system (pCSII-mVenus-hGeminin: 25-fold; ExEGFP-Lv105: 3,700-fold; pSMPUW-CMV-mRFP: 3,500-fold, FIG. A-C).FIG.3A-Dshows expression of EGFP in HEK293Ta cells 1 day after transfection of ExEGFP-Lv105 with the conventional third generation system or the PFL system. The higher expression of EGFP in the PFL system shows higher lentivirus production than conventional third generation system (FIG.4A-4D). To test potential functions of the transfer plasmid in the proposed PFL, the CMV and 5′LTR fusion promoter of pSMPUW-CMV-mRFP was changed to TRE and 5′LTR fusion promoter to enhance the transcription of genes sandwiched in between 5′ and 3′ LTRs (FIG.5A). However, the titer of lentiviruses made with TRE-5′LTR was similar as CMV-5′LTR (FIG.5B), which suggests that the virus gene expressions from helper plasmids were the rate-limiting step of lentivirus production and not the transfer plasmids. The titer of lentiviruses made with the PFL system is summarized on Table 1,FIG.7. The maximum titer observed was more than 1011TU/ml with ultracentrifuge concentration (pCSII-SYN-Venus; 1.4×1011±0.6×1011TU/ml).

Example 6

Development of a Safer Novel Packaging System

The helper plasmids used in this PFL system are second generation, and contain all HIV genomic DNA except env, vpr, and LTRs. To reduce the risks of handling of the PFL system, the rev and negative regulatory factor gene (net) were genetically excised from pPTK (TRE-ΔR8.2-Δvpr,rev,nef), and rev was cloned into pTRE-Tight vector under the control of TRE promoter (TRE-rev,FIG.2D). Thus, the above packaging system having extra safety features includes five helper plasmids. The titers of the lentiviruses made with the five helper plasmids were identical to the titers of the lentiviruses made with the four helper plasmids (FIG.2E). Similar to the four plasmids system, removing the TRE-tTA from system reduced the productivity of lentiviruses to 1,000 times (FIG.2E). Importantly, the lentiviruses produced in the PFL system was implemented in vivo. The lentivirus vector which carries synapsin promoter regulated Venus genes (Nagai, T. et al.Nat. Biotech.20, 87-90 (2002), incorporated herein by this reference) was generated with the four plasmid system and injected into the mouse left cerebral cortex (FIGS.6A and6B). The expression of Venus was shown in neurons from cortical layer two to five (FIGS.6C and6D). The lentivirus was also implemented to express cell cycle sensor mCherry-hGeminin gene (Sakaue-Sawano, A. et al.Cell132, 487-498 (2008), incorporated herein by this reference) in ex-vivo adult stem cell culture (FIG.4E-4G).