Patent Application
20220204946
The present invention relates to the development of modified envelope glycoproteins to pseudo-type viruses of the retroviridae family. These are derived from Murine leukaemia virus amphotropic, Gibbon Ape leukaemia virus and feline endogenous virus envelopes. The improved envelope glycoproteins contain, among other modifications, newly introduced alternative cleavable sequences.
The viral vectors pseudo-typed with these modified envelopes may be suitably employed for cargo delivery such as in gene and cell therapy applications, for the ex vivo and in vivo delivery of gene(s), protein(s), or molecule(s) of interest to a variety of target cells.
The modified envelope glycoproteins to pseudo-type viruses can be utilised using transient co-transfection of plasmids system or to develop stable cell lines producing recombinant viruses.
Therefore, the present invention is in the area of genetic engineering, diagnose, pharmaceutic and medical to be applied in medical human healthcare such as in gene and cell therapy applications, for the ex vivo and in vivo delivery of gene(s), protein(s), or molecule(s) of interest to a variety of target cells. Notwithstanding, it can be also used as a research tool or for veterinary and other applications.
The use of a highly pathogenic human virus for cargo delivery in therapeutic applications raises serious biosafety concerns. Therefore, the design of packaging systems evolved in order to increment the efficiency and the safety of retroviridae vectors while minimizing the possibility of replication-competent viruses during vector production.
Currently, several viral vectors derived from retroviridae virus family were developed to be used as therapeutics. Belonging to different genus of retroviridae are Alpharetrovirus, Betaretrovirus, Gammaretrovirus, Deltaretrovirus, Epsilonretrovirus, Lentivirus and Spumavirus. From the later, lentivirus derived vectors are currently the ones presenting the highest rate of utilization in clinical trials and have been approved to be used in the clinic.
Four generations of lentiviral vectors are considered. The first generation, developed by Naldini and co-workers, (Naldini, L. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263-7 (1996)) consisted in a three expression cassettes system. The packaging cassette had all structural, accessory and regulatory proteins, with the exception of the envelope glycoprotein. The transgene cassette was composed by the 5′LTR, the packaging signal, the RRE cis-acting region and the transgene under the control of a heterologous promoter. In the envelope glycoprotein expression cassette, the native HIV-1 envelope glycoprotein was replaced by the vesicular stomatitis virus G glycoprotein (VSV-G). With this system, good titers were easily achieved, but the poor safety level could not be accepted for a human and potentially lethal pathogen. Replication competent lentiviruses (RCL) could be generated with three homologous recombination events between the viral sequences of the packaging and transgene cassette or endogenous retroviral sequences within the producer cells. Additionally, the presence of the LTR promoter could activate neighbouring cellular genes, and the presence of all accessory HIV-1 genes, which are incorporated into the viral particle, enhanced the immunogenicity of the vector.
In the second generation, all accessory genes were deleted from the three plasmid system, without negative effects on vector titer. By removing the accessory genes (vpr, vif, vpu and nef), the generation of RCL became less probable and, if generated, would be unlikely to be pathogenic.
The third and still most widely used generation was developed by Dull and co-workers (Dull, T. et al. A third-generation lentivirus vector with a conditional packaging system. J. Virol. 72, 8463-71 (1998)). It is characterized by the deletion of the tat gene from the packaging plasmid and rev gene placed in an independent plasmid. By the use a chimeric 5′LTR with a heterologous viral promoter/enhancer, such as those of Cytomegalovirus (CMV) or Rous sarcoma virus (RSV). Therefore, lentiviral vector expression is independent of Tat. Rev is maintained but is provided by an independent non-overlapping plasmid.
Thus, this system has a total of four constructs, increasing the number of homologous recombination events required for RCL formation.
Another feature of this generation is the partial deletion of the 3′LTR in the transgene cassette, leading to transcriptional inactivation of the LTR promoter, after reverse transcription. These vectors are called self-inactivating (SIN) vectors.
This inactivation increases safety and reduces concerns related to insertional mutagenesis in the neighbouring sequences that can lead to the transactivation or up-regulation of neighbouring genome sequences, such as oncogenes.
A fourth generation of lentiviral vectors, Rev-independent, has also been developed by means of replacing RRE with heterologous viral sequences or by codon-optimization. These packaging systems are not, however, easily available for the research community. Also, the reported titers are one to two logs lower than that of the second or third generation systems.
Lentiviral vectors can be produced by transient transfection or stable producer cell lines. In transient transfection production, cells are co-transfected with the viral constructs, for example in the third generation system it is necessary to provide: (1) the viral vector genome transgene plasmid, (2) gag-pol helper plasmid, (3) Rev helper plasmid and (4) the plasmid expressing the envelope glycoprotein. Between 24 to 72 hours post-transfection, the lentiviral vectors present in the supernatant are harvested.
Stable production relies on packaging cell lines (PCLs) in which all of the components necessary to produce vectors are integrated into the cells' genome. In the scope of the present invention also semi-stable productions were performed. In this procedure only one, two or three of the above mentioned plasmids are stably integrated in the cell and therefore the productions of lentiviral vectors is performed by transiently transfecting these cells with three, two or one of the remaining necessary plasmids respectively.
The envelope glycoprotein used to pseudo-type the viral vectors defines the tropism of the virus by interacting with specific cell surface proteins and promoting the entrance of viruses into the host cell. The natural tropism of HIV-1 envelope glycoprotein is restricted to CD4+ cells, thus limiting its gene therapy applications to CD4+ cells like macrophages or T cells. However, lentiviral vectors have the ability to incorporate in their viral particles envelope glycoproteins from other viruses. This feature, denominated pseudo-typing, allows the manipulation of vector tropism.
The most used envelope glycoprotein for pseudo-typing lentiviral vectors is VSV-G, due to its wide tropism (possibly pantropism), high titres provided and improved vector stability, allowing the concentration of the particles by ultracentrifugation and resistance to freeze-thaw cycles. Despite all the advantages, VSV-G is toxic to producer cells, posing a challenge for stable production of lentiviral vectors pseudo-typed with this envelope glycoprotein.
Moreover, the broad tropism of VSV-G can be an impediment for targeted transduction of specific tissues, for example, for in vivo applications. Another limitation to its use for in vivo application is the inactivation of VSV-G by human complement present in the blood.
Several alternative envelope glycoproteins have been studied and are also suitable for pseudo-typing lentiviral vectors, for example, the amphotropic Murine leukaemia virus (MLV) 4070A envelope glycoprotein which is able to transduce most cells.
Other envelope glycoproteins have been engineered to pseudo-type lentiviral vectors with increased efficiency, for example the chimeric envelope glycoproteins RD114A and RDpro derived from the endogenous Feline leukaemia virus (RD114) and GaLV10A1 derived from the Gibbon ape leukaemia virus (GaLV).
Despite specific advantages and disadvantages, each envelope glycoprotein confers a different set of properties to the lentiviral vector, and so each pseudo-type may have its own potential niche.
The present invention aims to develop a tool, in particular an envelope glycoprotein for retroviridae viral vector pseudo-typing (including lentiviral vectors) that allows to overcome the problems mentioned above of the prior art by enhancing viral titres by modifying the cleavage site of the HIV-1 protease in the TM subunit resulting in a product with reduced toxicity when compared to VSV-G envelope.
The present invention relates to the development of a modified envelope glycoproteins to pseudo-type viruses of the retroviridae family. These are derived from Murine leukaemia virus amphotropic, Gibbon Ape leukaemia virus and feline endogenous virus envelopes. The improved envelope glycoproteins contain, among other modifications, newly introduced alternative cleavable sequences.
For this purpose, mutant Gammaretroviruses envelope glycoproteins based on 4070A, RD114A and GaLV10A1 are described and in order to enhance viral titers the R peptide region and or cleavage site of the HIV-1 protease in the TM subunit were engineered.
In addition to wild type lentiviral protease a mutated version of the HIV-1 protease is also described. This mutation is referred as leading to a 5- to 10-fold decrease in the protease activity compared to the WT HIV-1 protease and an expected, reduced cytotoxicity without effecting virus maturation and infectivity.
In summary novel chimeric envelope glycoproteins were engineered at the cytoplasmatic tail. All envelope variants developed showed to be incorporated in retroviral particles being suitable for viral vector pseudotyping and thus in cargo delivery. The envelope proteins modified from 4070A and RD114A can be used alternatively to the original counterparts to provide higher titers when using less active retroviral proteases. The novel GaLV10A1 derived glycoproteins can be used to improve viral titers when using WT or less active viral proteases such as T26S viral protein.
The increase in virus yields obtained, reduces the required amount of material required for clinical applications, and therefore reduce its costs and the complexity of such procedures.
A) Expression vectors for lentiviral genome, Gag-Pro-Pol, Rev and VSV-G envelope expression (from top to bottom).
B) Expression vectors developed for 4070A envelope and their derivatives.
C) Expression vectors developed for RD114A and their derivatives.
D) Expression vectors developed for GalVA and their derivatives.
Abbreviations: CMV, Cytomegalovirus promoter; RSV, Rous Sarcoma Virus promoter; hPGK, human phosphoglycerate kinase promoter; Int, intron; GFP, green fluorescence protein; GPP, gag-pro-pol sequence; GP(T26S)P, gag-pro-pol with mutated T26S protease sequence; Ψ, packaging signal sequence; WPRE, woodchuck hepatitis posttranscriptional regulatory element; pAn, polyA sequence; ZeoR, zeocin resistance gene; VSV-G, glycoprotein G of the vesicular stomatitis virus; resistance gene.
On top are represented the surface and transmembrane subunits and domains of the envelope glycoproteins: ectodomain, transmembrane domain (TMD) and cytoplasmic tail (CT).
The blue, sky blue, green and orange bars represent sequences from 4070A, 10A1, RD114 and GaLV envelope glycoproteins, respectively.
The square outlined in black is the R-peptide sequence.
The white, yellow and red lightning bolt shapes represent the non-modified, pro and giflet mutations on the protease cleavage site of the R-peptide, respectively. *: original envelope glycoproteins.
Representative pictures taken during transient transfection of 293T cells with the envelope glycoproteins 4070A, RD114 and GaLV and their derivatives as indicated in the photographs legends. 80× Bright field microscopy. Scale bar: 45 μm.
The bars correspond to infectious particles and the dots to total particles. The numbers on the top of the bars indicate fold increase of infectious titer relatively to the corresponding non modified envelope glycoprotein.
All values are shown as average±standard deviation of three biological replicates (n=3). Fold increase is shown for significant changes based on a one-tailed non-paired t-test, *p<0.1 and **p<0.01. Detection limit of 4.0×104 I.P./ml is indicated by a dashed arrow. WT: wild-type HIV-1 protease; T26S: T26S HIV-1 protease.
The present invention relates to the development of modified envelope glycoproteins to pseudo-type viruses of the retroviridae family and to the obtained envelope glycoproteins. The improved envelope glycoproteins contain, among other modifications, newly introduced alternative cleavable sequences.
The viral vectors pseudo-typed with these modified envelopes may be suitably employed for cargo delivery such as in gene and cell therapy applications, for the ex vivo and in vivo delivery of gene(s), protein(s), or molecule(s) of interest to a variety of target cells.
1. Process for Generating Modified Envelope Glycoproteins
The modified envelope glycoproteins to pseudo-type viruses of the retroviridae family are derived from Murine leukaemia virus amphotropic, Gibbon Ape leukaemia virus and feline endogenous virus envelopes. The improved envelope glycoproteins contain, among other modifications, newly introduced alternative cleavable sequences and can be utilised using transient co-transfection of plasmids system or to develop stable cell lines producing recombinant viruses.
Therefore, mutant Gammaretroviruses envelope glycoproteins based on 4070A, RD114A and GaLV10A1 are described and in order to enhance viral titres the R peptide region and or cleavage site of the HIV-1 protease in the TM subunit were engineered as described below.
2. Lentiviral Vectors Production
Lentiviral vectors can be produced by transient transfection or stable producer cell lines. In transient transfection production, cells are co-transfected with the viral constructs, for example in the third generation system it is necessary to provide: (1) the viral vector genome transgene plasmid, (2) gag-pol helper plasmid, (3) Rev helper plasmid and (4) the plasmid expressing the envelope glycoprotein. Between 24 to 72 hours post-transfection, the lentiviral vectors present in the supernatant are harvested.
In stable cell lines all the plasmids above are stably integrated in a mammalian cell line generating a producer cell which produces continuously lentiviral vectors.
In addition, two stable and transient production lentiviral vectors can be produced in semi-stable mode. In the latter case one (up to three) constructs mentioned above are stably integrated and productions can be carried out by providing transiently the remaining constructs.
2.1 Lentiviral Vectors Production by Transient Transfection
The generation of the constructs codifying the envelopes are described below.
Schematic representations of the constructed plasmids main transcriptional units are provided in
The transient transfection production of lentiviral vectors is performed for example from a third generation lentiviral packaging system by transfection of plasmid DNA, comprising:
2.2 Lentiviral Vectors Production by Stable Producer Cell Lines
Stable production relies on packaging cell lines (PCLs) in which all of the components necessary to produce vectors are integrated into the cells' genome.
In short, the process of the present invention comprises the production of a Gammaretrovirus modified envelope glycoproteins for vector pseudo-typing viruses of the retroviridae family derived from Murine leukaemia virus amphotropic, Gibbon Ape leukaemia virus or feline endogenous virus envelopes by modifying the transmembrane TM unit, altering its processing (i.e. cleavage), said process comprising the production of lentiviral vectors by stable production, wherein:
In a preferred embodiment of the invention, the stable production of lentiviral vectors is performed from for example a third generation lentiviral packaging system stably integrated in a producer cell line containing:
In exceptional cases the production can be carried out in semi-stable productions where some, but not all, constructs can be provided in transient to a cell stably expressing the remaining constructs.
3. Modified Envelope Glycoproteins
The most used envelope glycoprotein for pseudo-typing lentiviral vectors is VSV-G, due to its wide tropism, high titres provided and improved vector stability. However, VSV-G is toxic to producer cells. Therefore, the present invention proposes an alternative modified envelope glycoprotein for pseudo-typing lentiviral vectors that provides high titres and potentially improved vector stability, allowing the concentration of the particles by ultracentrifugation and resistance to freeze-thaw cycles and is safe.
These envelope glycoproteins are based on envelope glycoproteins 4070A derived from the murine leukaemia virus, RD114A derived from the endogenous Feline leukaemia virus and GaLV10A1 derived from the Gibbon ape leukaemia virus (GaLV) but modified in order to enhance viral titres through improved processing the TM subunit.
In result of this, modified sequences from 4070A, RD114 and GaLV envelope glycoproteins were produced with mutations on the TM namely on the protease cleavage site of the R-peptide by introduction of alternative cleavable sequences.
A short sequence—R-peptide—is cleaved from the cytoplasmic tail of retroviral envelope glycoproteins. Enhanced cleavage is expected when (i) homologous cleavage sequences, in relation to the viral protease, are used and (ii) highly active proteases are employed. The influence of the protease cleavage sequence on the R-peptide cleavage site and its impact on viral particles production was evaluated.
To this end, cleavage sites specifically recognizable by the HIV-1 protease were introduced in 4070A, RD114A and GaLV10A1. These envelope glycoproteins shared the retroviral cleavage site—VQAL↓VLTQ—of the cytoplasmic tail of 4070A. Herein, several envelope glycoproteins chimeras were constructed, engineered at the protease cleavage site of the R-peptide, to contain cleavage sequences recognized by HIV-1 proteolytic processing. These new glycoproteins were compared with their counterparts harbouring a murine leukaemia virus cleavage sequence.
For each envelope glycoprotein three mutations were performed:
Schematic representations of the constructed chimeric modified glycoproteins are provided in
4. Viral Vectors
Several viral vectors derived from retroviridae virus family were developed, in particular lentivirus derived vectors comprising the modified envelope glycoproteins based on 4070A, RD114A and GaLV10A1 according to the present invention were developed such as:
pMDLg/pRRET26S and pMDLg/pRRED25N are in-house constructed plasmids derived from pMDLg/pRRE, with the mutations T26S and D25N in the HIV-1 protease active site, respectively. The T26S mutation is described to cause reduced proteolytic activity and loss of protease-mediated cytotoxicity; the D25N mutation inactivates the active site of the protease. Also pRRLSIN-CMV-GFP plasmid was constructed derived from pRRLSIN.cPPT.PGK-GFP.WPRE driving the expression of enhanced green fluorescent protein (eGFP) from the CMV promoter.
5. Cell Lines Expressing the Modified Envelope Glycoproteins
Cell lines expressing the different glycoproteins described previously can be obtained by stable viral vector production. Stable production relies on packaging cell lines (PCLs) in which all of the components necessary to produce vectors are integrated into the cells' genome.
For transient production of lentiviral vectors, the third generation lentiviral packaging system and the transfection procedure as described in Tomás et al. (2013 and 2018) were used (Tomás, H. A., Rodrigues, A. F., Alves, P. M. Coroadinha, A. S. Lentiviral Gene Therapy Vectors: Challenges and Future Directions. Gene Therapy—Tools and Potential Applications (ed. Martin, F.). InTech (2013) pp. 287-317), (Tomás HA, Rodrigues A F, Carrondo M J T, Coroadinha A S. LentiPro26: novel stable cell lines for constitutive lentiviral vector production. Sci Rep. 8(1):5271. 2018).
The transfection procedure was conducted using PEI. HEK 293T cells were seeded at 5×104 cells/cm2 in 25 cm2 t-flask 24 h prior to transfection. A total of 4.65 μg of plasmid DNA per million cells was used for the transfection of one t-flask: 1 μg of pMDLg/pRRE or its variants (T26S and D25N) and 0.25 μg of pRSV-Rev (providing the packaging functions), 2.5 μg of pRRLSIN-CMV-GFP (providing the transgene vector) and 0.9 μg of plasmid codifying the envelope glycoprotein. After 20 to 24 hours post transfection, the medium was replaced with 4 ml of DMEM supplemented with 10% (v/v) FBS.
To assess transfection efficiency, transfected cells were harvested and analyzed for GFP fluorescence by flow cytometry (CyFlow® Space, Sysmex Corporation, Kobe, Japan).
pMDLg/pRRET26S and pMDLg/pRRED25N are in-house constructed plasmids derived from pMDLg/pRRE, with the mutations T26S and D25N in the HIV-1 protease active site, respectively. The T26S mutation is described to cause reduced proteolytic activity and loss of protease-mediated cytotoxicity; the D25N mutation inactivates the active site of the protease.
pRRLSIN-CMV-GFP is a third generation lentiviral transgene plasmid, driving the expression of enhanced green fluorescent protein (eGFP) from the CMV promoter. This plasmid is an in-house constructed plasmid, derived from pRRLSIN.cPPT.PGK-GFP.WPRE, as described by Dull, T. et al. (Dull, T. et al. A third-generation lentivirus vector with a conditional packaging system. J. Virol. 72, 8463-71. 1998) where the human phosphoglycerate kinase 1 (PGK) promoter was replaced by the CMV promoter.
Schematic representations of the plasmids main transcriptional units are provided in
Physical (total) particles (T.P.) in the serum-free viral supernatant were assessed by Nanoparticle Tracking Analysis (NTA) using NanoSight® NS500 (Malvern Instruments Ltd, Malvern, UK), following the manufacturer's instructions.
For titration of total particles by nanoparticle tracking analysis, serum-free DMEM was used in this step. After an additional production period of 24 hours, the medium containing the viral vectors was harvested, filtered through 0.45 μm-pore-size cellulose acetate filter for clarification, aliquoted and stored at −80° C. until further use.
For viral supernatants containing serum, a p24 enzyme-linked immunosorvent assay (ELISA)—INNOTEST HIV Antigen mAb (Fujirebio Diagnostics, Inc., Malvern, Pa., USA)—was used to quantify total particles, according to the manufacturer's instructions.
For titration of infectious particles (I.P.), HEK 293T cells were seeded at 5×104 cells/cm2 in 24-well plates 24 hours before infection. Transduction was performed by removing the cell supernatant and infecting cells with 0.2 ml of viral supernatants at several dilutions performed in DMEM supplemented with 10% (v/v) FBS containing 8 μg/ml of polybrene (Sigma-Aldrich).
For normal transduction protocol, cells were incubated at 37° C. overnight after which 0.5 ml of DMEM supplemented with 10% (v/v) was added. For spin inoculation protocol, the plates were centrifuged at 1200×g, 25° C. for 2 hours after which 0.5 ml of fresh supplemented DMEM was added and cells were incubated at 37° C. Two days after infection, cells were harvested and analyzed for GFP fluorescence by flow cytometry (CyFlow® Space).
The I.P. titer was determined taking into account the percentage of GFP positive cells, the number of cells determined at infection time and the dilution factor of the viral supernatant. Infections that rendered 2-20% of infected cells were considered for titer calculations.
Gammaretrovirus envelope glycoproteins, unlike VSV-G, undergo proteolytic processing during virion assembly mediated by the retroviral protease. A short sequence—R-peptide—is cleaved from the cytoplasmic tail, as described by Tedbury, P. R. & Freed, E. O. (Tedbury, P. R. & Freed, E. O. The cytoplasmic tail of retroviral envelope glycoproteins. Prog. Mol. Biol. Transl. Sci. 129, 253-84. 2015). This cleavage is required for virus entry, since it activates the fusogenic activity of the envelope glycoprotein.
The R-peptide cleavage site in the original envelope glycoproteins is specifically recognized by the retroviral protease. The efficiency of cleavage is dependent on both the sequence of cleavage and the protease used (i.e. its virus family origin and introduced mutations). Enhanced cleavage is expected when (i) homologous cleavage sequences, in relation to the viral protease, are used and (ii) highly active proteases are employed. The influence of the protease cleavage sequence on the R-peptide cleavage site and its impact on viral particles production was evaluated. To this end, cleavage sites specifically recognizable by the HIV-1 protease were introduced in 4070A, RD114A and GaLV10A1. These envelope glycoproteins shared the retroviral cleavage site—VQAL↓VLTQ—of the cytoplasmic tail of 4070A. Herein, several envelope glycoproteins chimeras were constructed, engineered at the protease cleavage site of the R-peptide, to contain cleavage sequences recognized by HIV-1 proteolytic processing. These new glycoproteins were compared with their counterparts harbouring a murine leukaemia virus cleavage sequence.
In addition, for each envelope glycoprotein three mutations were performed:
Schematic representations of the constructed chimeric modified glycoproteins are provided in
The engineered envelope glycoproteins were evaluated in transient production of lentiviral vector using the wild type (WT) and the mutated (T26S) HIV-1 proteases. The productions were assessed for both total particles, quantified by p24 ELISA, and infectious particles.
Mutations in the R-peptide cleavage site were found to mildly affect the infectious titers of lentiviral vectors pseudotyped with 4070A glycoprotein variants. RD114A modifications impacted the titers both negatively (RD114AΔR) and positively)(RD114Apro) when compared to the original counterpart. For GaLV10A1 derived glycoproteins, all mutations increased viral titers. Remarkably, infectious particles yields improvements up to 37-fold were observed for T26S HIV protease. Also an effect was also observed for WT HIV-1 protease, with a 5-fold improvement.
| Number | Date | Country | Kind |
|---|---|---|---|
| 115529 | May 2019 | PT | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2020/054804 | 5/20/2020 | WO | 00 |
