Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

1. System Design and Virus Production

1. Plasmid design and construction
   NOTE: The construction of an all-in-one LV-gRNA-dCas9-DNMT3A vector is performed by using a production-and expression-optimized expression cassette published by Ortinski et al.³⁰. The vector cassette carries a repeat of the recognition site of transcription factor Sp1 and a state-of-the-art deletion within the untranslated (U3') region of a 3'-long terminal repeat (LTR) (Figure 2A)^30,36. The vector backbone has been found to be effective in delivering and expressing CRISPR/Cas9^30,35.
   1. Obtain the deactivate (dead) version of SpCas9 (dCas9) via site-directed mutagenesis (data not shown). Replace the clone harboring D10A and H840A mutations in HNH and RuvC catalytic domains of the enzyme, respectively, with the active Cas9 in pBK301²⁹ by exchanging between AgeI-BamHI fragments (Figure 3).
   2. Derive the DNMT3A catalytic domain from pdCas9-DNMT3A-eGFP (see Table of Materials) by amplifying the DNMT3A portion BamHI-429/R 5'-GAGCGGATCCCCCTCCCG-3' BamHI-429/L 5'-CTCTCCACTGCCGGATCCGG-3' (Figure 3). To amplify the region containing DNMT3A, use the following conditions: (1) 95 °C for 60 s, (2) 95 °C for 10 s, (3) 60 °C for 20 s, (4) 68 °C for 60 s. Repeat conditions 2 to 4 30x. For the final extension, use 68 °C for 3 min and hold 4 °C.
   3. Clone the DNMT3A fragment, digested by a BamHI restriction enzyme, into the BamHI site of the modified pBK301 vector carrying dCas9. Verify the cloning by direct Sanger sequencing. Note that the resulted plasmid harbors dCas9-DNMT3A-p2a-puromycin transgene. The plasmid expresses gRNA scaffold from human U6 promoter (Figure 3).
   4. Replace the puromycin reporter gene with green fluorescent protein (GFP) to create dCas9-DNMT3A-p2a-GFP. Digest dCas9-DNMT3A-p2a-Puro plasmid with FseI. Purify the vector fragment using a gel purification method. Prepare the insert by digesting pBK201a (pLenti-GFP) with FseI. Clone the FseI fragment into the vector. The resulted plasmid pBK539 harbors dCas9-DNMT3A-p2a-GFP transgene (Figure 3).
2. Culturing HEK-293T cells and plating cells for transfection
   NOTE: Human embryonic kidney 293T (HEK-293T) cells are cultured in complete high-glucose Dulbecco’s modified Eagle’s medium (DMEM; 10% bovine calf serum, 1x antibiotic-antimycotic, 1x sodium pyruvate, 1x nonessential amino acid, 2 mM L-glutamine) at 37 °C with 5% CO₂. For the reproducibility of the protocol, it is recommended to test the calf serum when switching to a different lot/batch. Up to six 15 cm plates are needed for lentiviral production.
   1. Use low-passage cells to start a new culture (lower than passage 20). Once the cells reach 90%–95% confluence, aspirate the media and gently wash it with sterile 1x phosphate-buffered saline (PBS).
   2. Add 2 mL of trypsin-EDTA (0.05%) and incubate it at 37 °C for 3–5 min. To inactivate the dissociation reagent, add 8 mL of complete high-glucose DMEM, and pipette 10x–15x with a 10 mL serological pipette to create a single-cell suspension of 4 x 10⁶ cells/mL.
   3. For the transfections, coat 15 cm plates with 0.2% gelatin. Add 22.5 mL of high-glucose medium and seed the cells by adding 2.5 mL of cell suspension (total ~1 x 10⁷ cells/plate). Incubate the plates at 37 °C with 5% CO₂ until 70%–80% confluence is reached.
3. Transfection of HEK-293T cells
   1. Prepare 2x BES-buffered solution BBS and 1 M CaCl₂, according to Vijayraghavan and Kantor³⁵. Filter the solutions by passing them through a 0.22 µm filter and store them at 4 °C. The transfection mix has to be clear prior to its addition to the cells. If the mix becomes cloudy during incubation, prepare fresh 2x BBS (pH = 6.95).
   2. To prepare the plasmid mix, use the four plasmids as listed (the following mix is sufficient for one 15 cm plate): 37.5 µg of the CRISPR/dCas9-transfer vector (pBK492 [DNMT3A-Puro-NO-gRNA] or pBK539 [DNMT3A-GFP-NO-gRNA]); 25 µg of pBK240 (psPAX2); 12.5 µg of pMD2.G; 6.25 µg of pRSV-rev (Figure 4A). Calculate the volume of the plasmids based on the concentrations and add the required quantities into a 15 mL conical tube. Add 312.5 µL of 1 M CaCl₂ and bring the final volume to 1.25 mL, using sterile dd-H₂O. Gently add 1.25 mL of 2x BBS solution while vortexing the mix. Incubate for 30 min at room temperature. The cells are ready for transfection once they are 70%–80% confluent.
   3. Aspirate the media and replace it with 22.5 mL of freshly prepared high-glucose DMEM without serum. Add 2.5 mL of the transfection mixture dropwise to each 15 cm plate. Swirl the plates and incubate at 37 °C with 5% CO₂ for 2–3 h.
   4. After 3 h, add 2.5 mL (10%) of serum per plate and incubate overnight at 37 °C with 5% CO₂.
   5. On day 1 after the transfection, observe the cells to ensure that there is no or minimal cell death and that the cells formed a confluent culture (100%).
      1. Change the media by adding 25 mL of freshly prepared high-glucose DMEM and 10% serum to each plate.
   6. Incubate at 37 °C with 5% CO₂ for 48 h.
4. Harvest of the virus
   1. Collect the supernatant from all the transfected cells and pool them in 50 mL conical tubes. Centrifuge at 400–450 x g for 10 min. Filter the supernatant through a 0.45 µm vacuum filter unit. After filtration, the supernatant can be kept at 4 °C for short-term storage (up to 4 days). For long-term storage, prepare aliquots and store them at -80 °C.
      NOTE: The nonconcentrated viral preparations are expected to be ~2 x 10⁷ to 3 x 10⁷ vu/mL (refer to section 1.5 for titer determination). It is highly recommended to prepare single-use aliquots, since multiple freeze-thaw cycles will result in a 10%–20% loss in functional titers.
5. Concentration of viral particles
   NOTE: For the purification, a two-step double-sucrose method involving a sucrose gradient step and a sucrose cushion step is performed (Figure 4B).
   1. To create a sucrose gradient, prepare the conical ultracentrifugation tubes in the following order: 0.5 mL of 70% sucrose in 1x PBS, 0.5 mL of 60% sucrose in DMEM, 1 mL of 30% sucrose in DMEM, and 2 mL of 20% sucrose in 1x PBS.
   2. Carefully, add the supernatant, collected according to section 1.4, to the gradient. Since the total volume collected from four 15 cm plates is 100 mL, use six ultracentrifugation tubes to process the viral supernatant.
   3. Equally distribute the viral supernatant among each ultracentrifugation tube. To avoid tube breakage during centrifugation, fill the ultracentrifugation tubes to at least three-fourth of their total volume capacity. Balance the tubes with 1x PBS. Centrifuge the samples at 70,000 x g for 2 h at 17 °C.
      NOTE: To maintain the sucrose layer during the acceleration and deceleration steps, allow the ultracentrifuge to slowly accelerate and decelerate the rotor from 0 to 200 x g and from 200 to 0 x g during the first and last 3 min of the spin, respectively.
   4. Gently collect 30%–60% sucrose fractions into clean tubes (Figure 4B). Add 1x PBS (cold) up to 100 mL of total volume. Mix by pipetting multiple times.
   5. Carefully, stratify the viral preparation on a sucrose cushion by adding 4 mL of 20% sucrose (in 1x PBS) to the tube. Continue by pipetting ~20–25 mL of the viral solution per each tube. Fill the tubes with 1x PBS if the volume of their contents is less than three-fourth per tube. Carefully balance the tubes. Centrifuge at 70,000 x g for 2 h at 17 °C. Empty the supernatant and invert the tubes on paper towels to allow the remaining liquid to drain.
   6. Remove all the liquid by cautiously aspirating the remaining liquid. Note that, at this step, pellets containing the virus are barely visible as small translucent spots. Add 70 µL of 1x PBS to the first tube to resuspend the pellet. Thoroughly pipette the suspension and transfer it to the next tube until all pellets are resuspended.
   7. Wash the tubes with an additional 50 µL of 1x PBS and mix as before. Note that, at this step, the volume of the final suspension is ~120 µL and appears slightly milky. To obtain a clear suspension, proceed with a 60 s centrifugation at 10,000 x g. Transfer the supernatant to a new tube, make 5 µL aliquots, and store them at -80 °C.
      ​NOTE: Lentiviral vector preparations are sensitive to the repeated cycles of freezing and thawing. In addition, it is suggested that the remaining steps are done in tissue culture containment or in designated areas qualified in terms of being at adequate levels of biosafety standards (Figure 4B).
6. Quantification of viral titers
   NOTE: The estimation of viral titers is performed using the p24 enzyme-linked immunosorbent assay (ELISA) method (p24^gag ELISA) and according to the National Institutes of Health (NIH) AIDS Vaccine Program protocol for the HIV-1 p24 Antigen Capture Assay with slight modifications.
   1. Use 200 µL of 0.05% Tween 20 in cold 1x PBS (PBS-T) to wash the wells of a 96-well plate 3x.
   2. To coat the plate, use 100 µL of monoclonal anti-p24 antibody diluted at 1:1,500 in 1x PBS. Incubate the plate overnight at 4 °C.
   3. Prepare blocking reagent (1% bovine serum albumin [BSA] in 1x PBS) and add 200 µL to each well to avoid nonspecific binding. Use 200 µL of PBS-T to wash the well 3x for at least 1 h at room temperature.
   4. Proceed with the sample preparation. When working with concentrated vector preparations, dilute the vector at 1:100 by using 1 µL of the sample, 89 µL of dd-H₂0, and 10 µL of Triton X-100 (at a final concentration of 10%). For nonconcentrated preparations, dilute the samples at 1:10.
   5. Obtain HIV-1 standards by using a twofold serial dilution (the starting concentration is 5 ng/mL).
   6. Dilute the concentrated samples (prepared in step 1.6.4) in RPMI 1640 supplemented with 0.2% Tween 20 and 1% BSA to obtain 1:10,000, 1:50,000, and 1:250,000 dilutions. Similarly, dilute the nonconcentrated samples (prepared in step 1.6.4) in RPMI 1640 supplemented with 0.2% Tween 20 and 1% BSA to establish 1:500, 1:2,500, and 1:12,500 dilutions.
   7. Add the samples and standards on the plate in triplicates. Incubate overnight at 4 °C.
   8. The next day, wash the wells 6x.
   9. Add 100 µL of polyclonal rabbit anti-p24 antibody, diluted at 1:1,000 in RPMI 1640, 10% fetal bovine serum (FBS), 0.25% BSA, and 2% normal mouse serum (NMS), and incubate at 37 °C for 4 h.
   10. Wash the wells 6x. Add goat anti-rabbit horseradish peroxidase IgG diluted at 1:10,000 in RPMI 1640 supplemented with 5% normal goat serum, 2% NMS, 0.25% BSA, and 0.01% Tween 20. Incubate at 37 °C for 1 h.
   11. Wash the wells 6x. Add TMB peroxidase substrate and incubate at room temperature for 15 min.
   12. To stop the reaction, add 100 µL of 1 N HCl. In a microplate reader, measure the absorbance at 450 nm.
7. Measurement of fluorescent reporter intensity
   1. Use the viral suspension to obtain a 10-fold serial dilution (from 10^-1 to 10^-5) in 1x PBS.
   2. Plate 5 x 10⁵ HEK-293T cells in each well of a 6-well plate. Apply 10 µL of each viral dilution to the cells and incubate at 37 °C with 5% CO₂ for 48 h.
   3. Proceed to the fluorescence-activated cell sorting (FACS) analysis as follows: detach cells by adding 200 µL of 0.05% trypsin-EDTA solution. Incubate the cells at 37 °C for 5 min and resuspend them in 2 mL of DMEM medium (with serum). Collect the samples into a 15 mL conical tube and centrifuge at 400 x g at 4 °C. Resuspend the pellet in 500 µL of cold 1x PBS.
   4. Fix the cells by adding 500 µL of 4% paraformaldehyde (PFA) and incubate for 10 min at room temperature.
   5. Centrifuge at 400 x g at 4 °C and resuspend the pellet in 1 mL of 1x PBS. Analyze the GFP expression using a FACS instrument, as described in Ortinski et al.³⁰. To determine the virus functional titer, use the following formula.
      
      Here,
      Tg = number of GFP-positive cells;
      Tn = total number of cells;
      N = total number of transduced cells;
      V = volume used for transduction (in microliters).
8. Counting GFP-positive cells
   NOTE: Determine the multiplicity of infection (MOI) that is employed for transduction. Test a wide range of MOIs (from MOI = 1 to MOI = 10).
   1. Seed 3 x 10⁵ to 4 x 10⁵ HEK-293T cells per each well of a 6-well plate.
   2. When the cells reach >80% confluence, transduce them with the vector at the MOI of interest.
   3. Incubate at 37 °C with 5% CO₂, and monitor the GFP signal in the cells for 1–7 days.
   4. Count the number of GFP-positive cells. Employ a fluorescent microscope (Plan 4x objective, 0.1 N.A., 40x magnification) using a GFP filter (excitation wavelength = 470 nm, emission wavelength = 525 nm). Use untransduced cells to set the control population of GFP-negative cells.
   5. Employ the following formula to determine the functional titer of the virus.
      
      Here,
      N = number of GFP-positive cells;
      D = dilution factor;
      M = magnification factor;
      V = volume of virus used for transduction.
      NOTE: Calculate the results following this example: for 10 GFP-positive cells (N) counted at a dilution (D) of 10^-4 (1:10,000) at 20x magnification (M) in a 10 µL sample (V), the TU per milliliter will be (10 x 10⁴) x (20) x (10) x (100) = 2 x 10⁸ vu/mL.

2. Differentiation of dopaminergic neural progenitor cells

1. Culturing hiPSCs
   NOTE: hiPSCs from a patient with the triplication of the SNCA locus, ND34391, were obtained from the National Institute of Neurological Disorders and Stroke (NINDS) catalog (see Table of Materials).
   1. Culture hiPSCs under feeder-independent conditions in feeder-free ESC-iPSC culture medium (see Table of Materials) onto hESC-qualified basic matrix membrane (BMM)-coated plates (see Table of Materials). Wash confluent colonies with 1 mL of DMEM/F12, add 1 mL of dissociation reagent (see Table of Materials), and incubate for 3 min at room temperature.
   2. Aspirate the dissociation reagent and add 1 mL of feeder-free ESC-iPSC culture medium.
   3. Scrape the plate using a cell lifter and resuspend the colonies in 11 mL of feeder-free ESC-iPSC culture medium by pipetting 4x–5x using borosilicate pipettes.
   4. Plate 2 mL of colony suspension onto BMM-coated plates and place the plate at 37 °C with 5% CO₂. Perform a daily medium change and split the cells every 5–7 days.
2. Differentiation into dopaminergic neural progenitor cells
   NOTE: The differentiation of hiPSCs into dopaminergic neural progenitor cells (MD NPCs) is performed using a commercially available neural induction medium protocol per manufacturers’ instructions, with slight modifications^31,32 (see Table of Materials). The first day of the differentiation is considered as day 0. High-quality hiPSCs are required for efficient neural differentiation. The induction of MD NPCs is performedusing an embryoid body (EB)-based protocol.
   1. Prior to starting the differentiation of hiPSCs, prepare a microwell culture plate (see Table of Materials) according to its manufacturer’s instructions.
   2. After preparing the microwell culture plate, add 1 mL of neural induction medium (NIM; see Table of Materials) supplemented with 10 µM Y-27632.
   3. Set the plate aside until ready to use.
   4. Wash hiPSCs with DMEM/F12, add 1 mL of cell detachment solution (see Table of Materials), and incubate for 5 min at 37 °C with 5% CO₂.
   5. Resuspend single cells in DMEM/F12 and centrifuge them at 300 x g for 5 min.
   6. Carefully aspirate the supernatant and resuspend the cells in NIM + 10 µM Y-27632 to obtain a final concentration of 3 x 10⁶ cells/mL.
   7. Add 1 mL of the single-cell suspension to a single well of the microwell culture plate and centrifuge the plate at 100 x g for 3 min.
   8. Examine the plate under the microscope to ensure an even distribution of the cells among the microwell, and incubate the cells at 37 °C with 5% CO₂.
   9. On days 1–4, perform a daily partial medium change.
   10. Using a 1 mL micropipette, remove 1.5 mL of the medium and discard. Slowly, add 1.5 mL of fresh NIM without Y-27632.
   11. Repeat step 2.2.10 until day 4.
   12. On day 5, coat one well of a 6-well plate with BMM.
   13. Place a 37 µm reversible strainer (see Table of Materials) on top of a 50 mL conical tube (waste). Point the arrow of the reversible strainer upward.
   14. Remove the medium from the microwell culture plate without disturbing the formed EBs.
   15. Add 1 mL of DMEM/F12 and promptly collect the EBs with the borosilicate pipette and filter them through the strainer.
   16. Repeat step 2.2.15 until all EBs are removed from the microwell culture plate.
   17. Invert the strainer over a new 50 mL conical tube and add 2 mL of NIM to collect all the EBs.
   18. Plate 2 mL of the EB suspension into a single well of the BMM-coated plate using a borosilicate pipette. Incubate the EBs at 37 °C with 5% CO₂.
   19. On day 6, prepare 2 mL of NIM + 200 ng/mL SHH (see Table of Materials) and perform a daily medium change.
   20. On day 8, examine the percentage of neuronal induction.
   21. Count all attached EBs and, specifically, determine the number of each individual EB that is filled with neural rosettes. Quantify the neural rosette induction using the following formula.
       
       NOTE: If the neural induction is <75%, neural rosette selection may be inefficient.
   22. On day 12, prepare 250 mL of N2B27 medium with 119 mL of neurobasal medium, 119 mL of DMEM/F12 medium, 2.5 mL of GlutaMAX, 2.5 mL of NEAA, 2.5 mL of N2 supplement, 5 mL of B27 without vitamin A, 250 μL of Gentamicin (50 mg/mL), and 19.66 μL of BSA (7 mg/mL).
   23. To prepare 50 mL of complete N2B27 medium, add 3 μM CHIR99021, 2 μM SB431542, 20 ng/mL bFGF, 20 ng/mL EGF, and 200 ng/mL SHH.
       NOTE: It is important to prepare the completed medium right before use.
   24. Aspirate medium from the wells containing the neural rosettes and wash with 1 mL of DMEM/F12.
   25. Ad 1 mL of neural rosette selection reagent (see Table of Materials) and incubate at 37 °C with 5% CO₂ for 1 h.
   26. Remove the selection reagent and, using a 1 mL pipettor, aim directly at the rosette clusters.
   27. Add the suspension to a 15 mL conical tube and repeat steps 2.2.25 and 2.2.26 until the majority of the neural rosette clusters have been collected.
       NOTE: To avoid contamination with nonneuronal cell types, do not overselect.
   28. Centrifuge the rosette suspension at 350 x g for 5 min. Aspirate the supernatant and resuspend the neural rosettes in N2B27 + 200 ng/mL SHH. Add the neural rosette suspension to a BMM-coated well and incubate the plate at 37 °C with 5% CO₂.
   29. On days 13–17, perform a daily medium change using completed N2B27 medium. Passage the cells when the cultures are 80%–90% confluent.
   30. To split the cells, prepare a BMM-coated plate.
   31. Wash the cells with 1 mL of DMEM/F12, aspirate the medium, and add 1 mL of dissociation reagent (see Table of Materials).
   32. Incubate for 5 min at 37 °C, add 1 mL of DMEM/F12, and dislodge attached cells by pipetting up and down. Collect the NPC suspension in a 15 mL conical tube. Centrifuge at 300 x g for 5 min.
   33. Aspirate the supernatant and resuspend the cells in 1 mL of complete N2B27 + 200 ng/mL SHH.
   34. Count the cells and plate them at a density of 1.25 x 10⁵ cells/cm², and incubate the cells at 37 °C with 5% CO₂.
   35. Change the medium every other day, using complete N2B27 + 200 ng/mL SHH.
       NOTE: At this passage, NPCs are considered as passage (P) 0. SHH can be withdrawn from the N2B27 medium at P2.
   36. Passage the cells once they reach 80%–90% confluence.
   37. At this stage, confirm that the cells express Nestin and FoxA2 markers by using immunocytochemistry and qPCR. This protocol leads to the generation of 85% double-positive cells for the Nestin and FoxA2 markers.
   38. For passaging cells, repeat steps 2.2.31–2.2.36.
   39. Freeze the cells, starting from passage P2. For the freezing of cells, repeat steps 2.2.31–2.2.36 and resuspend the cell pellet at 2 x 10⁶ to 4 x 10⁶ cells/mL using cold neural progenitor freezing medium (see Table of Materials).
   40. Transfer 1 mL of the cell suspension into each cryovial and freeze the cells using a standard slow-rate-controlled cooling system. For long-term storage, keep the cells in liquid nitrogen.
3. Thawing MD NPCs
   1. Prepare a BMM-coated plate and warm complete N2B27. Add 10 mL of warm DMEM/F12 to a 15 mL conical tube. Place a cryovial in a 37 °C heat block for 2 min.
      1. Transfer the cells from the cryovial to the tube containing DMEM/F12. Centrifuge at 300 x g for 5 min.
      2. Aspirate the supernatant, resuspend the cells in 2 mL of N2B27, and add the cell suspension to one well of the BMM-coated plate. Incubate the cells at 37 °C with 5% CO₂.

3. Transduction of MD NPCs and the analysis of methylation changes

1. Transduction of MD NPCs
   1. Transduce MD NPCs at 70% confluence with LV-gRNA/dCas9-DNMT3A vectors at MOI = 2. Replace the N2B27 medium 16 h posttransduction.
   2. Add N2B27 with 5 µg/mL puromycin, 48 h after the transduction. Culture the cells for 3 weeks into N2B27 plus puromycin to obtain the stable MD NPC lines. Cells are ready for downstream applications (DNA, RNA, protein analyses, phenotypic characterization²³, freezing and passaging).
2. Characterization of the methylation profile of SNCA intron 1
   1. Extract DNA from each stably transduced cell line using a DNA extraction kit (see Table of Materials).
   2. Use 800 ng of DNA to perform a bisulfite conversion, using a commercially available kit (see Table of Materials). After the bisulfite conversion, elute the bisulfite-converted DNA to 20 ng/µL.
3. PCR for the pyrosequencing analysis
   1. Prepare the PCR master mix in a nuclease-free tube. For each reaction, use 0.4 µL of reverse primer (10 µM), 0.4 µL of forward primer (10 µM), 1.6 µL of MgCl₂ (25mM), 2 µL of 10x CoralLoad Concentrate, 10 µL of 2x PCR master mix, 4 µL of 5x Q-Solution, 1 µL of DNA, and 0.6 µL of nuclease-free water.
   2. Transfer the reaction plate to a thermocycler and perform PCR using the following conditions: 95 °C for 15 min, 50 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 30 s, with a final 10 min extension step at 72 °C. Primers used for the pyrosequencing of SNCA intron 1 are listed in Table 1, Figure 7A, and Supplementary Figure 1.
   3. After amplification, visualize the amplicons using 2 µL of PCR product with ethidium bromide staining, following agarose gel electrophoresis.
   4. Pyrosequencing assays are validated by using mixtures of unmethylated (U) and methylated (M) bisulfite-converted DNAs in the following ratios, namely 100U:0M, 75U:25M, 50U:50M, 25U:75M, and 0U:100M (see Table of Materials).
   5. Conduct pyrosequencing using pyrosequencing reagents (see Table of Materials), and calculate the methylation values for each CpG site using pyrosequencing software. For a detailed pyrosequencing protocol, refer to Bassil et al.³⁷.