METHOD OF TRANSFECTING MACROPHAGES

Abstract

The development of the method of the invention enables the efficient and reproducible production of genetically modified GMP-grade human macrophages. The inventors have described the effects of the method described on cell viability and efficiency of introduction of genetic material into macrophages. Unlike previous methods of introducing genetic material into macrophages, excellent conditions are demonstrated which produce efficient transgene expression, without compromising cell viability. Critically, the method of the invention does not use virus to introduce genetic material, is efficacious on mature cells, are functional with in vitro assay and in vivo transfer in a liver disease model and complies with practices compatible with manufacture and delivery of these cells to patients.

Claims

1. A method of transfecting a human macrophage with genetic material, the method comprising the steps of: (a) contacting a human macrophage with genetic material; (b) electroporating the macrophage with a first pulse phase, wherein the first pulse phase comprises a burst of unipolar pulses or a square pulse, wherein each pulse is between 750-1000V, and wherein the first pulse phase lasts for a total period of between 20-500 .Math.s; and (c) electroporating the macrophage with a second pulse phase, wherein the second pulse phase comprises a burst of pulses or a square pulse, wherein each pulse is between 50-225 V, and wherein the second pulse phase lasts for a total period of 2000-50000 .Math.s.

2. A method according to claim 1, wherein each pulse in the first pulse phase is selected from: i) between 800-1000 V; ii) between 850-1000 V; iii) between 900-1000 V; iv) between 950-1000 V; or v) about 950 V.

3. A method according to any preceding claim, wherein the first pulse phase is for a total period of time selected from: i) between 40-180 .Math.s; ii) between 80-120 .Math.s; or iii) about 120 .Math.s.

4. A method according to any preceding claim, wherein the first pulse phase comprises a burst of unipolar pulses and the second pulse phase comprises a burst of unipolar pulses.

5. A method according to any preceding claim, wherein each pulse in the second pulse phase is selected from: i) between 80-200 V; ii) between 85-175 V; iii) between 90-150 V; iv) between 95-125 V; or v) about 100-125 V.

6. A method according to any preceding claim, wherein the second pulse phase is for a total period of time selected from: i) between 11000-30000 .Math.s; ii) between 12000-30000 .Math.s; iii) between 11000-25000 .Math.s; iv) between 12000-25000 .Math.s; v) between 11000-23000 .Math.s; vi) between 12000-23000 .Math.s; or vii) about 23000 .Math.s.

7. A method according to any preceding claim, wherein the burst of unipolar pulses are positive or negative pulses.

8. A method according to any preceding claim, wherein the genetic material comprises a nucleic acid, preferably DNA or RNA.

9. A method according to any preceding claim, wherein the genetic material comprises one or more nucleic acids encoding one or more genes of interest and/or regulatory elements.

10. A method according to any preceding claim, wherein the genetic material comprises one or more nucleic acids encoding a homologous or heterologous gene of interest, preferably a heterologous gene of interest.

11. A method according to any preceding claim, wherein the genetic material is comprised on a vector, preferably a plasmid.

12. A method according to any preceding claim where the human macrophages are human monocyte derived macrophages.

13. A method according to any preceding claim, wherein the human macrophage is contacted with genetic material in solution, preferably the solution is conductive, preferably the solution is an electroporation buffer.

14. A method according to claim 13, wherein the human macrophage is present in the solution at a cell density selected from: i) between 1×10.sup.5 to 1×10.sup.9cells/mL; ii) between 5×10.sup.5 to 8×10.sup.8 cells/mL: iii) between 1×10.sup.6 to 6×10.sup.8 cells/mL; iv) between 5×10.sup.6 to 5×10.sup.8 cells/mL; v) between 5×10.sup.7 to 1.5×10.sup.8/mL; vi) between 1×10.sup.7/mL to 1×10.sup.9/mL or v) 5×10.sup.7 cells/mL.

15. A method according to any of claims 13 or 14, wherein the genetic material is present in the solution at a concentration selected from: i) between 1 to 10 .Math.g per 5×10.sup.6 macrophage cells; ii) between 3 to 9 .Math.g per 5×10.sup.6 macrophage cells; iii) between 4 to 8 .Math.g per 5×10.sup.6 macrophage cells; or iv) between 5 to 7.5 .Math.g per 5×10.sup.6 macrophage cells.

16. A transfected human macrophage produced by the method of any of claims 1-15.

17. A transfected human macrophage comprising a heterologous nucleic acid, wherein the macrophage has a repressed STING pathway and optionally wherein the macrophage has reduced expression of IFN-β.

18. A transfected human macrophage according to claim 17, wherein the macrophage is polarized.

19. A population of transfected human macrophages according to claims 16 to 18.

20. A population of transfected human macrophages comprising a viability selected from of at least: (i) 60%; ii) 70%; iii) 80%; iv) 85%; v) 90%; or vi) 95%, and wherein said population of macrophages optionally comprises a heterologous nucleic acid.

21. A population of transfected macrophages according to claim 20, wherein the macrophages have a repressed STING pathway, optionally wherein the macrophages have reduced expression of IFN-β.

22. A transfected human macrophage according to claim 17, or a population of transfected human macrophages according to claim 19, wherein the macrophage is non-virally transfected.

23. A transfected human macrophage according to claims 16 or 17, or a population of transfected human macrophages according to claims 19 or 20, for use as a medicament.

24. A transfected human macrophage or population thereof for use according to claim 23, in the treatment of a fibrotic disease, optionally a fibrotic liver disease.

25. A transfected human macrophage or a population thereof for use according to claim 23 or 24, wherein the human macrophage is autologous or allogenic to the donor.

Description

FIGURES

[0368] FIG. 1 shows: Optimisation of electroporation first pulse parameters: Flow cytometry was used to assess viability (DRAQ-7 negative), efficiency (GFP+ cells) and intensity of expression of GFP (GFP mean) from the pMax-GFP vector 24 hours post-electroporation using unique first pulse parameters with a second low-voltage burst pulse (125 V, 8 .Math.sec burst length for 23000 .Math.sec), measured using Miltenyi MACSQuant flow cytometer. *p<0.05, **p<0.01 one-way ANOVA with Tuckey’s post-test (n=4 per group) (A) Comparison of viability, efficiency and expression intensity of pMax-GFP electroporation with different first pulse modes (square pulse, burst unipolar pulse or burst bipolar pulse). (B) Assessment of viability, efficiency and expression intensity of pMax-GFP with different first pulse voltages with a burst unipolar mode. (C) Assessment of viability and efficiency of pMax-GFP with different first pulse lengths with a burst unipolar mode, and different first pulse burst lengths with a burst unipolar mode measured with an Acea Novocyte flow cytometer. D) Assessment of viability and efficiency of pMax-GFP with single pulse in different modes measured with an Acea Novocyte flow cytometer. Scale bars represent 200 .Math.m.

[0369] FIG. 2 shows: Optimisation of second pulse parameters: Flow cytometry was used to assess viability (DRAQ-7 negative), efficiency (GFP+ cells) and intensity of expression of GFP (GFP mean) from the pMax-GFP vector 24 hours post-electroporation using unique second pulse parameters measured using Miltenyi MACSQuant flow cytometer. (A) Comparison of viability, efficiency and expression intensity of pMax-GFP electroporation with different combinations of first pulse (square pulse or burst unipolar pulse) and second pulse modes (square pulse or burst pulse). (B) Assessment of viability, efficiency and expression intensity of pMax-GFP with different second pulse voltages with a burst unipolar (first pulse) and burst (second pulse) configuration. (C) Assessment of viability, efficiency and expression intensity of pMax-GFP with different second pulse lengths with a burst unipolar (first pulse) and burst (second pulse) configuration. (D) Assessment of viability and efficiency of pMax-GFP with different second pulse burst lengths with a burst unipolar (first pulse) and burst (second pulse) configuration.

[0370] FIG. 3 shows: Assessment of transfection efficiency with different amounts of plasmid: Flow cytometry was used to assess viability (DRAQ-7 negative), efficiency (GFP+ cells) and intensity of expression of GFP (GFP mean) from the pMax-GFP vector 24 hours post-electroporation measured using Miltenyi MACSQuant flow cytometer, testing (A) different amounts of pMax-GFP vector in 5-day differentiated macrophages. (B) Viability, efficiency and intensity was compared with different amounts of pMax-GFP vector in 5 versus 7-day differentiated macrophages.

[0371] FIG. 4 shows: Reproducibility of optimised transfection conditions: Flow cytometry was used to assess viability (DRAQ-7 negative) and efficiency (GFP+ cells) of expression of GFP from the pMax-GFP vector 24 hours post-electroporation using a 950 V first pulse in burst unipolar mode, combined with a burst second pulse of 100 V, 125 V (ideal conditions) and 150 V (non-ideal control). For ideal conditions, n=3 to 14 unique donor macrophages. (B) Phase/contrast microscopy shows healthy morphology of macrophages. Combined data from Miltenyi MACSQuant and Acea Novocyte flow cytometers. Scale bars represent 200 .Math.m.

[0372] FIG. 5 shows: Maintenance of macrophage function in vitro and in vivo with post-transfection and STING inhibitor treatment: (A) Analysis of secreted interferon (IFN) proteins in hMDMs electroporated with up to 10 .Math.g pMax-GFP vector or electroporated without the presence of DNA (mock). (B) Analysis of secreted IFNβ protein in untransfected (UT), mock transfected (mock) or GFP transfected (GFP) hMDMs and cultured post-transfection with either M-CSF (control) or polarising factors IFNy, IFNy+LPS, IL4+IL13 or IL10 (C) Phase/contrast microscopy shows healthy morphology of macrophages using ideal conditions and treatment with STING inhibitor BX-795 versus DMSO control. (D) In vitro phagocytosis assay of untransfected hMDMs (control) versus GFP-transfected macrophages treated with BX-795 (GFP + BX-795), with M2-like polarising factors (GFP + BX-795 + IL4/IL13 or IL10) or DMSO control (GFP). (E) Serum liver function tests and histological analysis of picrosirius red (PSR) staining of collagen in chronic CCl.sub.4mice treated with cryopreserved hMDM (cryo), GFP-transfected hMDM treated with DMSO (GFP), GFP-transfected hMDM treated with BX-795 (GFP+B) or PBS vehicle (vehicle). *p<0.05 one-way ANOVA.

[0373] FIG. 6 shows: Improvement of STING inhibition and transfection cell density: (A) ELISA for IFN-b was used to dose the protein in cell culture s/n from macrophages transfected with GFP and treated for 16h with a combination of IL-4 and IL-13 at various concentration. Flow cytometry was used to determine the expression of GFP (GFP MFI) and the viability of macrophages transfected with GFP and treated for 16h with a combination of IL-4 and IL-13 at various concentration. In all graph, every connected series of symbols (open or filled) represent a distinct donor. (B) ELISA for IFN-b was used to dose the protein in cell culture s/n from macrophages transfected with GFP and treated for 16h with IL-10 at various concentration. Flow cytometry was used to determine the expression of GFP (GFP MFI) and the viability of macrophages transfected with GFP and treated for 16 h with IL-10 at various concentration. In all graph, every connected series of symbols represent a distinct donor. (C) Flow cytometry was used to quantify the expression of GFP (GFP MFI) and the viability of macrophages transfected with either GFP or a vector combining the expression of GFP or CCR2-GFP. In all graph, every symbol represents a distinct concentration of macrophages at the time of transfection, and the solid line connects data from the same donor. The same key applies for all graphs in FIG. 6C.

EXAMPLES

Materials and Methods

Generation and Characterization of Macrophages

[0374] Human monocyte-derived macrophages were generated from CD14+ peripheral blood monocytes as previously described [16]. Briefly, peripheral blood mononuclear cells were isolated from whole blood buffy coat material from healthy volunteer donors according to established protocols [16]. CD14+ monocytes were enriched using CD14 CliniMACS beads (Miltenyi). Purity and differentiation were assessed as previously described [16]. Monocytes were cultured in TexMACS-GMP medium (Miltenyi) supplemented with GMP-grade M-CSF (100 ng/mL; RnD Systems) for 5 or 7 days and differentiation assessed by flow cytometry. Monocytes were CD45+, CD14+, CD15-, 25F9-low, CD206-low. Macrophages were CD45+, CD14+, 25F9+ and CD206+.

Electroporation

[0375] Monocyte-derived macrophages were harvested, counted and resuspended at a density of 5 × 10.sup.7 cells/mL of CliniMACS Electroporation Buffer (Miltenyi). 25-100 .Math.g/mL of pMax-GFP plasmid (Lonza) was mixed with the cell suspension for electroporation. For electroporation, 100 .Math.l of cell/plasmid mix was introduced into 0.2 cm electroporation cuvettes and electroporation conducted with the CliniMACS Prodigy Electroporator (Miltenyi). Parameters assessed were 1st pulse voltage and type, and 2nd pulse voltage, type and length. Following electroporation, cells were cultured as above, with or without the STING inhibitor BX-795 (Invivogen) up to a concentration of 10 .Math.M.

Test of an Alternative Method to Inhibit STING Pathway

[0376] Following electroporation, 200 .Math.L sterile TexMACS was added to the cuvette and the cells were collected in a Falcon tube (pre-filled with 5 ml TexMACS) using a 18G needle attached to a 1 mL syringe. 500 .Math.L TexMACS were added to the cuvette to collect any remaining cells. The macrophage suspension was spun down at 300× g, 4° C. for 5 min. The supernatant was aspirated, and the cells were resuspended at concentration 4*10.sup.6 cells/mL in TexMACS supplemented with 100 ng/mL rh-M-CSF.

[0377] Macrophages were immediately treated with IL4 and IL13 (0, 10, 20 or 50 ng/mL). 10.sup.5 cells/well from each condition were seeded in a 96-well plate for flow cytometry/phagocytosis assessment. The remaining cells from each condition were seeded in 48-well plates for RNA analysis.

Optimisation of Cell Density at the Point of Electroporation

[0378] Day5 macrophages were spun down at 300*g, 4° C. for 5 min. The supernatant was discarded, and the cells were resuspended in electroporation buffer at the concentrations outlined below: [0379] 50×10.sup.6 cells/ml - 15×10.sup.6 cells in 300 .Math.l electroporation buffer (previously used cell density) [0380] 75×10.sup.6 cells/ml - 15×10.sup.6 cells in 200 .Math.l electroporation buffer [0381] 100×10.sup.6 cells/ml - 15×10.sup.6 cells in 150 .Math.l electroporation buffer [0382] 150×10.sup.6 cells/ml - 15×10.sup.6 cells in 100 .Math.l electroporation buffer

[0383] 5 .Math.g per 5×10.sup.6 cells of GFP-expressing plasmid was added to the transfected cells immediately prior to electroporation. After electroporation as per protocol above, transfection efficiency was evaluated by flow cytometry, and RNA was preserved for future RNA analysis.

Assessment of Transfection Efficiency by Flow Cytometry

[0384] 24 hours post-electroporation, cells were dissociated by pipetting up and down and a 200.Math.l aliquot placed in a flow cytometry tube for analysis. To assess viability, 1 .Math.l of DRAQ-7 (Abcam) viability dye was added to the cell suspension and the cells were assessed by flow cytometry on a MACSQuant flow cytometer (Miltenyi), or Novocyte flow cytometer (Acea). Viability was assessed as the proportion of DRAQ7-negative cells. GFP-positivity was assessed compared to untransfected controls and expressed as the proportion of GFP+ live (DRAQ-7-negative) cells. GFP intensity was assessed using mean fluorescence intensity.

Phagocytosis Assay

[0385] Cells were seeded in 96 well plates at a density of 200,000 per well in triplicate wells and left to adhere overnight. The following day, medium was aspirated and replaced with TexMACS medium containing NucBlue reagent (Invitrogen) and incubated for 30 min at 37° C. to stain nuclei. Medium was aspirated, cells washed in PBS, and then 100 .Math.l PBS was added to cells. 100 .Math.l of pHrodo E. coli red beads (diluted 1:10 in PBS) was added to the cells, and the cells were observed using an Operetta (PerkinElmer) to photograph fluorescence every 5 minutes over 145 minutes using a 40X objective. Images were analysed using Columbus. Nuclei were identified by NucBlue staining, and pHrodo red fluorescence intensity was measured in a cytoplasmic ring region around the nucleus. Fluorescence is reported as average pHrodo red fluorescence per nucleus.

Assessment of Secreted Proteins

[0386] Interferon proteins secreted_into the medium of hMDMs were analysed using a U-PLEX Human Interferon Bundle kit on a MESO Quickplex SQ 120 according to the manufacturers’ instructions (Meso Scale Discovery).

Mouse Experiments

[0387] NOD CB17 Prkdc/.sup.SCID mice were supplied by Charles River and housed in individually ventilated cages in a sterile animal facility with a 10-14-hours dark/light cycle and free access to food and water. All procedures were performed in accordance with UK Home Office guidelines (Animals [Scientific Procedures] Act 1986). Chronic liver fibrosis was induced in adult male mice over a 12-week period by twice weekly intraperitoneal injections of carbon tetrachloride (CCl.sub.4) dissolved in sterile olive oil at a concentration of 0.2 mL/kg for the first week increasing to 0.4 mL/kg for further 10 weeks. One day after the 18th CCl.sub.4 injection (9 weeks), mice were randomly allocated to receive either day5 cryopreserved (CP) hMDMs (n = 5), day 5 macrophages transfected with pMax-GFP vector and cultured for a further 24 hours (GFP; n=4), day 5 macrophages transfected with pMax-GFP vector and cultured for a further 24 hours in medium supplemented with 5 .Math.M BX-795 (GFP+B; n=4), or injected with saline (vehicle, n = 6) injections via tail vein. The intra-splenic route would have ensured maximal cell delivery, but it does not model the administration route used in the phase I MATCH trial (day7 hMDMs in patients with chronic liver fibrosis)[17]. All hMDMs were suspended in sterile saline at a density of 5 × 10.sup.7 cells/mL and 0.1 mL was injected via a 30-gauge needle (Myjector 0.3 mL syringes, Terumo). Injection of cells or saline was repeated at week 10 and week 11. 0.2 mL/kg CCl.sub.4 administration continued for an additional week.

[0388] All mice were culled at the indicated time points using anaesthesia overdose followed by cervical dislocation as confirmatory method. Organs and blood were retrieved, processed and stored for further analysis: liver left lobe was snap frozen and stored at -80° C.; the other liver lobes were fixed in formalin 10% for 8 h and then included in paraffin blocks; kidneys, spleen, heart and lungs were fixed in formalin 10% for 8 h and then included in paraffin blocks; blood was collected in Eppendorf, left to sediment for 8 h and then spun at 10000x g for 10 minutes at room temperature to obtain serum, to be stored at -80° C.; blood collected in EDTA-coated tubes (Microvette CB300, Sarstedt) were used to collect 30⍰L of full blood to use for the analysis of the haematological parameters using the CellTac machine (Nihon Kohden).

Liver Function Tests on Sera

[0389] Serum chemistry was performed by measurement of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin, and serum albumin. ALT was measured using a commercial kit (Alpha Laboratories Ltd). AST and ALP were determined by a commercial kit (Randox Laboratories). Total bilirubin was determined by the acid diazo method described by Pearlman and Lee [18] using a commercial kit (Alpha Laboratories Ltd). Mouse serum albumin measurements were determined using a commercial serum albumin kit (Alpha Laboratories Ltd). All kits were adapted for use on a Cobas Fara centrifugal analyzer (Roche Diagnostics Ltd). For all assays, intra-run precision was CV < 4%. In some experiments, assays were run on plasma samples with the exception of ALP activity.

Histological Analysis

[0390] Picrosirius red (PSR) staining was performed according to standard protocols. Morphometric pixel analysis to quantify histological staining was performed. For fibrosis quantification PSR stained section were scanned to create a single image with Polaris slide scanner (Perkin Elmer). A second scan on the same machine was performed to obtain multi-spectral image acquisition on 10 to 15 fields/slide at 10x magnification. Multi-spectral images were analysed using the Trainable WEKA Segmentation mode using the InForm software (Perkin Elmer).

Results

1. Optimisation of First Pulse Parameters

[0391] We assessed the parameters of the first pulse in combination with a second lower voltage pulse, or with a single pulse in different modes. Initially we assessed the type of pulse using pre-set conditions forT-cell electroporation as baseline conditions (950 V burst-bipolar first with burst second pulse). The types of first pulse assessed were the baseline ‘burst-bipolar’ mode, versus ‘burst-unipolar’ or′square’ pulse modes. We found that a first pulse in ‘burst-unipolar’ or ‘square’ mode provided similar transfection efficiencies (77.95 ± 7.44% for ‘burst-unipolar’ vs 76.12 ± 8.61% for ‘square’ pulse GFP+ cells) and viability (81.29 ± 6.11% for ‘burst-unipolar’ vs 71.11 ± 9.62% for ‘square’ pulse DRAQ7- cells) for macrophages, as measured by flow cytometry. Using a ‘burst-bipolar’ first pulse resulted in a lower transfection efficiency (61.11 ± 13.85% GFP+ cells) and adequate viability (84.59 ± 6.01% DRAQ7- cells; FIG. 1A). We next assessed the impact of first pulse voltage over the complete range (0 V - 1000 V) of the Prodigy electroporator with a ‘burst-unipolar’ first pulse mode. Electroporation efficiency increased exponentially up to 900 V, and plateaued between 900 V and 1000 V, without significant loss of cell viability (FIG. 1B). GFP expression was confirmed with fluorescence microscopy (not shown).

[0392] We next assessed the effects of first pulse length and burst length in burst-unipolar mode combined with a second low voltage burst pulse. Transfection efficiency increased up to 250 .Math.sec, plateauing between 80-250 .Math.sec, and viability decreased below 50% after 250 .Math.sec (FIG. 1D). Transfection efficiency increased with increased burst length, while cell viability was decreased with transfection, plateauing from 5-40 .Math.sec (FIG. 1C).

[0393] Having assessed a number of different first pulse types in combination with a second lower voltage pulse, we assessed efficiency and viability of macrophages subjected to a single electroporation pulse without a second lower voltage pulse. We observed low transfection efficiency (<20% GFP+ cells) with only a single pulse with square, burst-unipolar or burst-bipolar modes (FIG. 1D).

[0394] From these experiments, we determined that a dual pulse electroporation, with a first pulse of 900 V - 1000 V in ‘burst-unipolar’ mode with a burst length of 5-40 .Math.sec and a total pulse length of 80-250 .Math.sec was ideal for introduction of the pMax-GFP vector into macrophages with minimal loss of viability.

2. Optimisation of Second Pulse Parameters

[0395] We assessed the effect of second pulse parameters on transfection efficiency and viability. Firstly, pulse combinations of (‘1st pulse’ + ‘2nd pulse’) ‘burst-unipolar’ + ‘burst’, ‘burst-unipolar’ + ‘square’ and ‘square’ + ‘square’ were assessed. A ‘burst-unipolar’ first pulse + ‘burst’ second pulse gave the maximum viability (~90% live) and transfection efficiency (~94% GFP+). Although a ‘burst-unipolar’ first pulse + ‘square’ second pulse resulted in a high transfection efficiency (~85% GFP+), a considerable reduction in viability (~65% live) was observed. The combination of ‘square’ first pulse + ‘square’ second pulse resulted in the lowest transfection efficiency (~72% GFP+) and viability (.sup.~58% live; FIG. 2A). Using the optimised 950 V ‘burst-unipolar’ first pulse we next assessed the effect of second pulse voltage between 0 V and 300 V. Transfection efficiency increased exponentially up to 100 V, plateauing from 100 V- 200 V (~90% - 94% GFP+ cells), then decreasing sharply up to 300 V (.sup.~10% GFP+ cells). Viability remained high up to 125 V (>90% live), then decreased to ~58% as voltage increased to 300 V (FIG. 2B).

[0396] Finally, we assessed the effect of second pulse length over the range of 0 .Math.sec - 50000 .Math.sec. Efficiency increased exponentially up to 6000 .Math.sec, plateauing between 12000 .Math.sec and 50000 .Math.sec (.sup.~90% - 95% GFP+ cells). However, viability decreased as the length of the pulse was increased, plateauing at .sup.~90% from 12000 .Math.sec to 23000 .Math.sec, and reducing to .sup.~69% at 50000 .Math.sec (FIG. 2C). We also assessed the effect of second pulse burst length. We found that a second pulse burst length of 5-8 .Math.sec gave the greatest efficiency (66.16 - 82.66% GFP+) and viability (82.22 - 86.66% DRAQ-7 negative; FIG. 2D). From these experiments, we determined that a second pulse of 100 V - 125 V in ‘burst’ mode, with a burst length of 5-8 .Math.sec a time of 12000 - 30000 .Math.sec was ideal for introduction of the pMax-GFP vector into macrophages.

3. Assessment of the Effects of Macrophage Differentiation Protocols and Amount of DNA

[0397] We assessed the impact on electroporation efficiency with different amounts of plasmid DNA in macrophages differentiated using a 5 day ‘no feed’ protocol, or the standard 7 days with one feed protocol. Viability was not affected by the amount of plasmid DNA used for electroporation. However, we observed a decrease in efficiency and intensity with less than 5 .Math.g or more than 7.5 .Math.g of plasmid DNA (FIG. 3A). Compared to day 5 macrophages, day 7 macrophages cells had reduced viability, GFP+ cells and GFP intensity when electroporated under the same conditions (FIG. 3B).

4. Reproducibility of Macrophage Electroporation

[0398] Having determined ideal conditions for electroporation of primary human macrophages, we combined data from all electroporation experiments to evaluate the reproducibility of our method (up to 14 individual donors) using a first burst unipolar pulse of 950 V with a burst second pulse of 100 V or 125 V for 23000 .Math.sec as ‘ideal’ conditions for electroporation. A second pulse voltage of 150 V represented a ‘non-ideal’ condition for comparison. Although donor variation was observed, electroporation efficiency and viability was highly reproducible (FIG. 4A). Viability was high for ideal conditions (DRAQ-7-negative cells: 87.40 ± 4.92%; n=3 for 100 V, 79.40 ± 3.28%; n=14 for 125 V), with similar efficiency observed for both conditions (GFP-positive cells: 86.90 ± 4.28%; n=3 for 100 V, 84.15 ± 3.37%; n=14 for 125 V). Phase contrast microscopy showed healthy, intact morphology of macrophages 24 hours post-electroporation (FIG. 4B) and fluorescence microscopy demonstrated visible GFP expression in macrophages electroporated in the presence of pMax-GFP vector (not shown).

5. Maintenance of Post-Electroporation Macrophage Function by Inhibition of the Stimulator of Interferon Genes (STING) Pathway

[0399] Upregulation of interferon (IFN) genes by the cGAS-cGAMP-STING pathway is a feature of macrophages encountering foreign DNA, and potentially affects function. Therefore, in order for macrophage genetic modification to be efficacious, a method to overcome this innate sensing mechanism must be employed. We tested the activation of this pathway by assessing the secretion of IFN proteins following electroporation of hMDMs with pMax-GFP. We observed secretion of IFNα2α, IFNβ and IFNy with introduction of pMax-GFP vector into hMDMs (FIG. 5A). We also assessed secretion of STING pathway-induced IFNβ following electroporation in the presence or absence of the pMax-GFP vector (‘GFP’ or ‘mock’ respectively) compared to untransfected cells (‘UT’) and overnight culture with M1-like polarising stimuli (IFNy or IFNy+LPS) or M2-like polarising stimuli (IL4+IL13 or IL10). IFNβ secretion was induced by introduction of pMax-GFP into macrophage by electroporation, but not by electroporation alone. All polarising stimuli reduced the secretion of IFNβ, with IFNβ at undetectable control levels with the combination of IL4+IL13 in GFP-transfected cells (FIG. 5B). We utilised the STING inhibitor molecule BX-795 to inhibit this pathway and test in vitro function by measuring phagocytosis, and in vivo function by measuring the ability of cells to reduce liver fibrosis. Cells remained healthy and GFP-expressing up to 8 .Math.M of BX-795, with some loss of cells at 10 .Math.M (FIG. 5C). We measured phagocytic capacity of untransfected hMDMs or GFP-transfected hMDMs in the presence or absence of BX-795, and with BX-795 combined with polarization to M2-like alternatively activated cells with either IL4/IL13 or IL10. Phagocytic capacity, measured by the average fluorescence of pHRodo beads per cell, was approximately 41% of non-genetically modified hMDMs in GFP-transfected hMDMs treated with DMSO vehicle, and was recovered to approximately 66% of non-genetically modified hMDMs in GFP-transfected hMDMs treated with BX-795. Phagocytic capacity was not improved with the combination of M2-like polarising stimuli to BX-795 treatment (FIG. 5D). Mouse models of liver cirrhosis triggered by reiterative CCl.sub.4-induced hepatocyte injury are a useful tool to test the safety and efficacy of cell therapy product. The induction phase of liver cirrhosis commonly last 4 to 12 weeks, depending on the extent of fibrosis desired [19-21]. We envisage our cell therapy being used in cases of advanced fibrosis therefore we chose to treat our mice for 12 weeks with CCl.sub.4. Testing macrophage-based cell therapy products requires a xenotransplant of human cell into mice. To avoid rejection, we opted to use immunodeficient mice. However, because these mice lack an appropriate immune response to liver fibrosis, they are unlikely to benefit from the paracrine effect of macrophage cell therapy on the mouse own immune response.

[0400] In the present pilot experiment, we compared the injection of 1×10.sup.6 cryopreserved hMDMs, GFP-transfected hMDMs treated with DMSO vehicle or BX-795 at week 9, 10 and 11 of CCl.sub.4 treatment. Control mice are injected with an equivalent volume of saline only (vehicle). Mice are culled at week 12 and blood and organs collected for further analysis. Histological analysis of the quantity of fibrosis in the liver by PSR staining and quantification revealed an average decrease in fibrosis of 6.3% in cryopreserved hMDMs treated, and 8.5% in GFP-transfected hMDMs treated with BX-795 proportionally to saline treated mice. However, no difference was observed between GFP-transfected hMDMs treated with DMSO vehicle (FIG. 5E). Sera analysis confirmed a positive effect of the cryopreserved and GFP-transfected hMDMs treated with BX-795: Results show a trend towards a decrease in liver enzymes ALT and AST and, more importantly, a reduction in bilirubin circulating levels (FIG. 5E), one of the main factors in monitoring the results of macrophage cell therapy in the clinic [4]. No change in circulating GLDH and Albumin was noted.

[0401] These data show that the combination of introducing DNA into macrophages via electroporation with treatment with an inhibitor of the innate DNA sensing mechanism enables the production of genetically modified macrophages that are functional in vitro and in vivo and are safe for use in animal models of liver disease.

6. Maintenance of Post-Electroporation Macrophage Function by Inhibition of the Stimulator of Interferon Genes (STING) Pathway

[0402] Starting from the results expressed in FIG. 5B, we postulated that treatment of macrophages post-transfection with combination of IL4 and IL13 or IL10 could be used as an alternative strategy to inhibit the STING pathway, thereby improving macrophage phenotype and function.

[0403] To this end, we treated macrophages with 10 ng/mL, 20 ng/mL or 50 ng/mL of a combination of IL4 and IL13, and with 20 ng/mL or 50 ng/mL of IL10. We transfected macrophages with GFP, and we measured IFNβ production in cell culture supernatants after 16h from transfection (GFP), from electroporation only (mock) or from plating without transfection or electroporation (control). Treatment with IL4+IL13 resulted in a trend towards reduction of IFN-β production in both donors (control mean = 55.39 pg/mL; 10 ng/mL mean = 40.39 pg/mL; 20 ng/mL mean = 42.24 pg/mL; 50 ng/mL mean = 39.64 pg/mL) (FIG. 6A). Various concentration of IL4+IL13 induced only minor changes in GFP expression, with 20 ng/mL being the most conservative condition (GFP MFI; FIG. 6A; Control (0 ng/mL) GFP transfected = 12636; 10 ng/mL GFP transfected = 9909; 20 ng/mL GFP transfected = 11045; 50 ng/mL = 9226). Viability was also unaffected by IL4+IL13 treatment (Live %; FIG. 6A; Control (0 ng/mL) GFP transfected = 87.07%; 10 ng/mL GFP transfected = 83.34%; 20 ng/mL GFP transfected = 82%; 50 ng/mL = 85.88%).

[0404] We also treated macrophages with 20 ng/mL or 50 ng/mL of a combination of IL10, and with 20 ng/mL or 50 ng/mL of IL10. We transfected macrophages with GFP, and we measured IFNβ production in cell culture supernatants after 16h from transfection (GFP), from electroporation only (mock) or from plating without transfection or electroporation (control). Treatment with IL10 resulted in a trend towards reduction of IFN-β production only at a concentration of 50 ng/mL (control mean = 431.47 pg/mL; 20 ng/mL mean = 524.16 pg/mL; 50 ng/mL mean = 392.39 pg/mL) (FIG. 6B). Various concentration of IL10 had a slight effect in reducing GFP expression (GFP MFI; FIG. 6B; Control (0 ng/mL) GFP transfected = 786; 20 ng/mL GFP transfected = 418; 50 ng/mL = 531). Viability was largely unaffected by IL10 treatment (Live %; FIG. 6B; Control (0 ng/mL) GFP transfected = 78.69%; 20 ng/mL GFP transfected = 70.69%; 50 ng/mL = 70.36%).

[0405] We also optimised the electroporation concentration of macrophages. We compared 50×10.sup.6/mL (concentration used so far) with 75×10.sup.6/mL; 100×10.sup.6/mL; and 150×10.sup.6/mL. We transfected a plasmid expressing GFP (.sup.~3.5 kb) and we record results at 24 h and 48 h post-transfection. We also transfected a plasmid encoding for CCR2 and GFP (i.e. a larger plasmid, .sup.~6kb). We tested CCR2-GFP at 24 h and 48 h using 50×10.sup.6/mL (concentration used so far); 75×10.sup.6/mL; and 100×10.sup.6/mL, but not at 150 ×10.sup.6/mL, due to limitations in the available cell numbers. 75×10.sup.6/mL and 150×10.sup.6/mL offered a significant increase in GFP expression at 24 h and 48 h, using the GFP plasmid, as assessed by flow cytometry (FIG. 6C; see below for a table with the raw MFI values at 24 h). Viability was also unaffected by the concentration at the point of electroporation (FIG. 6C; see below for a table with the raw percentage values at 24 h). 75 ×10.sup.6/mL offered a significant increase in GFP expression at 24 h and 48 h, using the GFP-CCR2 plasmid (150 ×10.sup.6/mL not assessed -n.a.- using this plasmid), as measured by flow cytometry (FIG. 6C; see below for a table with the raw MFI values at 24 h). Viability was also unaffected by the concentration at the point of electroporation (FIG. 6C; see below for a table with the raw percentage values at 24 h).

TABLE-US-00001 50 ×10.sup.6/mL 75×10.sup.6/mL 100×10.sup.6/mL 150 ×10.sup.6/mL GFP CCR2-GFP GFP CCR2-GFP GFP CCR2-GFP GFP CCR2-GFP GFP MFI 1859591 1252692 3190158 1511828 2942530 1145144 3665920 n.a. Viability (%) 76.06 73.03 81.75 75.55 71.38 74.89 79.77 n.a. Raw percentage values at 24h post-transfection

[0406] Collectively, these data show that various STING inhibition strategy can be deployed without affecting transgene expression and macrophage viability. Finally, these data support the possibility of using a broad range of cell concentration at the point of electroporation, a feature useful when devising treatment courses with different cell dosages.

Conclusions

[0407] The development of the method of the invention enables the efficient and reproducible production of genetically modified GMP-grade human primary monocyte-derived macrophages. We have described the effects of different first and second pulse parameters on cell viability and efficiency of introduction of genetic material into macrophages. Unlike previous methods of introducing genetic material into macrophages, we demonstrate the ideal conditions which produce efficient transgene expression, without compromising cell viability, and our experiments provide a robust framework for the optimisation of introduction and expression of vectors encoding different genes of interest. Critically, the method of the invention does not use virus to introduce genetic material, is efficacious on mature cells, are functional with in vitro assay and in vivo transfer in a liver disease model and complies with practices compatible with manufacture and delivery of these cells to patients.

Equivalents

[0408] Those skilled in the art will recognise or be able to ascertain using no more than routine experimentation, equivalents of the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims. Any combination of the embodiments disclosed in the any plurality of the dependent claims or Examples is contemplated to be within the scope of the disclosure.

INCORPORATION BY REFERENCE

[0409] The disclosure of each and every patent, patent application publication, and scientific publication referred to herein is specifically incorporated herein by reference in its entirety, as are the contents of its Figures.

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