Construction of AAV vectors
crRNA expression vector design and construction
- Identify genes for knockout by targeted delivery of HDR template. Here we chose TRAC and PDCD1, but note that any gene with a Cpf1 PAM sequence may be targeted.
- Design LbCpf1 crRNA (20bp) with Benchling or other computational pipelines.
- Synthesize oligonucleotides with two LbCpf1 direct repeat and sticky ends.
Digest pXD017 with FD BbsI and insert guide after U6 promoter (pXD017-39).
CAR sequence generation
- Generation of CD22BBz CAR as previously described 11. CD22 binding scFV (m971) specific for the human CD22 followed by CD8 hinge-transmembrane-regions linked to 4-1BB (CD137) intracellular domains and CD3ζ intracellular domain.
- The sequence of CD19 binding scFv (FMC63) was found from NCBI (GenBank: HM852952) and followed by CD8 hinge-transmembrane-regions linked to 4-1BB (CD137) intracellular domains and CD3ζ intracellular domain 12. In order to detect CD19BBz CAR in different way, the Flag-tag sequence (GATTACAAAGACGATGACGATAAG) was added after the CD8⍺ leader sequence.
Synthesize m971-BBz and FMC63-BBz using gBlock (IDT).
HDR template design
- Amplify left and right homologous arms of the TRAC or PDCD1 locus from primary CD4+ T cells by PCR using locus-specific primer sets with multiple cloning site (MCS). PCR annealing temperature (60℃).
TRAC_HDR_F1 (With AAV vector overlap sequence) TCAACTAGATCTTGAGACAAGGTACGATGTAAGGAGCTGCTGTGACT
TRAC_HDR_R1 (With MCS) GGTACCTCGAGCGTACGGGTCAGGGTTCTGGATATCTGTG
TRAC_HDR_F2 (With MCS) CGTACGCTCGAGGTACCGAGAGACTCTAAATCCAGTGACAAG
TRAC_HDR_R2 (With AAV vector overlap sequence) CTTTTATTAAGCTTGATATCGAATTGTGGGTTAATGAGTGACTGCG
PDCD1 _HDR_F1 (With AAV vector overlap sequence) TGGCAGGAGAGGGCACGTGGGCAGCCTCACGTAGAAGGAA
PDCD1 _HDR_R1 (With MCS) TCCGAGAATTCTTTGTTAACTGTGTTGGAGAAGCTGCAGGT
PDCD1 _HDR_F2 (With MCS) CACAGTTAACAAAGAATTCTCGGAGAGCTTCGTGCTAAACTGG
PDCD1 _HDR_R2 (With AAV vector overlap sequence) GCGGCCGCTCGGTCCGCACCTGATCCTGTGCAGGAGGG
Sequence amplicons (Yale Keck or any other Sanger sequencing facility).
AAV-crRNA-HDR-CAR vector cloning
- pXD040 construction: Clone HDR sequences into the AAV vector (pXD017-39) by Gibson assembly. Incubate samples in a thermocycler at 50°C for 30 minutes.
pXD043 (CD22CAR) and pXD054 (CD19CAR) construction: Digest pXD040 with BsiWI and Acc65I, and then clone CAR sequences into MCS by Gibson assembly.
AAV production and titration
- Transfect HEK293FT cells with AAV constructs in 15-cm tissue culture dishes, AAV2 transgene vectors, packaging (pDF6) plasmid, and AAV6/9 serotype plasmid together with polyethyleneimine (PEI).
Collect transfected cells with PBS after 72 hours of transfection.
AAV purification and titration
- Mix transfected cells with pure chloroform (1/10 volume).
- Incubate cells at 37 °C with shake vigorously for 1 h.
- Add NaCl to a final concentration of 1 M.
- Centrifuge at 20,000g at 4 °C for 15 mins.
- Transfer aqueous layer to another tube and discard the chloroform layer.
- Add PEG8000 to sample until 10% (w/v) and shake until dissolved.
- Incubate mix at 4 °C for 1 h and then centrifuge at 20,000g at 4 °C.
- Discard supernatant and suspend pellet in DPBS with MgCl2.
- Treat sample with universal nuclease and incubate at 37 °C for 30 mins.
- Add chloroform (1:1 volume), shake, and centrifuge at 12,000g at 4°C for 15 mins.
- Isolate aqueous layer and concentrate through a 100-kDa MWCO. Critical step: concentrate AAV at high concentration so the volume can be reduced when performing the infection, which can decrease the toxicity of AAV. AAV should be aliquoted and stored at -80℃.
Titer virus by qPCR using custom Taqman assays (ThermoFisher) targeted to promoter U6.
T cell electroporation
- Human primary peripheral blood CD4+ T cells were acquired from healthy donors (STEMCELL technologies). T cells were cultured in X-VIVO media (Lonza) with 5% human AB serum and recombinant human IL-2 30U/mL.
- Activate T cells with CD3/CD28 Dynabeads for 2 days prior to electroporation.
- Use magnetic holder to remove Dynabeads.
- Prepare cells at a density of 2 x 105 cells per 10 μL tip reaction or 2 x 106 cells per 100 μL tip reaction in electroporation Buffer R (Neon Transfection System Kits).
- Mixed with 1 μg or 10 μg of modified NLS-LbCpf1-NLS mRNA (TriLink) according to reaction volume.
- Electric shocked at program 24 (1,600V, 10ms, and three pulses).
- Transfer cells into 200µl or 1mL of pre-warmed X-VIVO media (without antibiotics) immediately after electroporation.
Add indicated volumes of AAV (AAV volume to not exceed 20% of culture volume) into the T cells 2-4 hours after electroporation. CAR will begin to be expressed after two to three days and have enrichment after stimulation with target cells.
CAR-T detection by flow cytometry
- After electroporation for 5 days, incubate 1×106 CD22BBz CAR transduced T cells with 0.2 μg CD22-Fc (R&D system) in 100 μL PBS for 30 mins, and then stain with PE-IgG-Fc and FITC-CD3 antibodies for 30 mins.
- For CD19BBz CAR detection, incubate CD19BBz CAR transduced T cells with APC-anti-DYKDDDDK Tag and FITC-CD3 antibodies for 30 mins.
- Wash cells twice after staining. Quantify and sort labeled cells on BD FACSAria II.
The staining patterns were analyzed using FlowJo software 9.9.4 (Treestar, Ashland, OR).
- Five days after electroporation, harvest the bulk transduced T cells and sorted T cells. The genomic DNA was collected using the QuickExtract DNA Extraction Solution (Epicentre).
- PCR amplify target loci from genomic DNA around cutting site.
- Run PCR amplicons on 2% E-gel EX and purify (with known band size) using QIAquick Gel Extraction Kit.
- After purification, denature 200 ng of purified PCR product, anneal, and digest with T7E1, 37℃ 45min (New England BioLabs).
Load digested PCR products into 2% E-gel EX and quantify DNA fragment abundance using E-Gel™ Low Range Quantitative DNA Ladder (ThermoFisher).
HDR quantification and NGS sequencing analysis
Semi-quantitative In-Out PCR
- Use three primers for In-Out PCR:
TRAC 1st: binds to a sequence from the left TRAC homology arm
TRAC 2st: binds to genomic sequence outside of this AAV donor
CD22CAR 3rd: recognizes a sequence contained in the m971-BBz cassette
TRAC 1st: CCCTTGTCCATCACTGGCAT
TRAC 2st: GCACACCCCTCATCTGACTT
CD22CAR 3rd: GAAATCAAAGCGGCCGCAG
- Normalize amplicon (labeled TRAC-HDR) concentration by comparison to the product resulting from the uninfected control with genomic DNA isolated from human CD4+ T cells.
- PCR products were used for Nextera library preparation following the manufacturer’s protocols (Illumina).
Prepped libraries were sequenced using 100-bp paired-end reads on an Illumina HiSeq 4000 instrument or equivalent.
- Some PCR products from amplification around cutting site of genomic DNA (same samples as T7E1 assay) were used for Nextera library preparation following the manufacturer’s protocols (Illumina).
- Prepped libraries were sequenced using 100-bp paired-end reads on an Illumina HiSeq 4000 instrument or equivalent (generating 29 to 74 million reads per library).
- Map paired reads to amplicon sequences (expected sequences provided in FASTA form to generate indices) using BWA-MEM with the -M option.
- Discard 100bp reads in SAM file that fall outside a +/- 75bp window of expected cut site within the amplicon.
- Discard soft-clipped reads (identified with “S” character in CIGAR string).
- Identify indel reads by the presence of "I" or "D" characters within the CIGAR string.
Quantify cutting efficiency as percentage of indels over total (indel plus wild-type reads) within the defined window.
- Map reads to possible amplicons based on primer combinations and HDR status.
- Define "informative" amplicons as truncated so that 100bp reads would have at least 20bp homology with the CAR sequence (or with the other TRAC arm, in the case of wild-type sequences). Informative reads can be used to distinguish wild-type, NHEJ and HDR reads with higher confidence.
- Map paired reads to amplicon sequences using BWA-MEM with -M flag to generate SAM files.
- Use SAMtools to convert SAM files to BAM, sort, index, and generate summary statistics of read counts with the idxstats option.
- To quantify wild-type vs NHEJ reads, take reads that mapped to “info_nonHDR” sequence (described below), and call reads with indels (I" or "D" characters within the CIGAR string) as NHEJ. Otherwise call reads as wild-type.
- Pool read counts for downstream analysis.
Schema for our amplicon sequences and quantifications provided below:
amplicon_nonHDR: refers to full amplicon from F1 and R1 of genomic, wild-type DNA.
amplicon_CAR_F1: refers to full amplicon from F1 and R1 of expected, integrated CAR.
amplicon_CAR_F2: refers to full amplicon from F2 (primer site within the CAR as opposed to outside) and R1 of expected, integrated CAR.
info_nonHDR same as amplicon_nonHDR, except truncated to 80bp of the TRAC arms.
info_CAR_F1: same as amplicon_CAR_F1, except truncated to 80bp of the TRAC arms flanking the TRAC-CAR interface.
info_CAR_F2: same as amplicon_CAR_F2, except truncated to 80bp of the TRAC arms flanking the TRAC-CAR interface (relevant to the right arm only, since F2 is within the CAR sequence).
HDR, NHEJ, and WT scores were calculated as follows:
info_nonHDR = info_WT + info_NHEJ
hdr_score = info_CAR_F2/(info_CAR_F2+info_nonHDR)
wt_score = info_WT/(info_CAR_F2+info_nonHDR)
nhej_score = info_NHEJ/(info_CAR_F2+info_nonHDR)
Co-culture functional assays
Stable cell line generation
- Generate lentivirus including GFP-Luciferase reporter genes.
- Infect NALM6 cells (ATCC) with 2x concentrated lentivirus by spinoculation in retronectin-coated (Takara) plates at 800g for 45 mins at 32°C.
- After infection for 2 days, sort GFP positive cells (NALM6-GL) by flow cytometry.
- Perform a second round of sorting after culturing for an additional two days.
Incubate cells with 150g/ml D-Luciferin (PerkinElmer) and measure bioluminescence signal intensity by an IVIS system to assess luciferase expression.
Cancer cell cytolytic assay (kill assay)
- Seed 2104 NALM6-GL cells in a 96 well plate.
- Co-culture modified T cells with NALM6-GL at indicated E:T ratios for 24 hours.
Add 150g/ml D-Luciferin (PerkinElmer) into each well and measure luciferase assay intensity by a plate reader (PerkinElmer) to assess cell proliferation.
T cell exhaustion assay
- Co-culture T cells modified by AAV with NALM6-GL cells at 0.5:1 E:T ratio for 24 hours.
- Collect cells and wash once by DPBS. Incubate cells with 0.2 μg CD22-Fc (R&D Systems) in 100 μL DPBS for 30 mins.
- Stain cells with PE-IgG-Fc, PD-1-FITC, TIGIT-APC and LAG3-Percp/cy5.5 (Biolegend) for 30 mins.
Measure stained cells by flow cytometry.
Intracellular staining of IFNγ and TNF-
- After infection for 5 days, co-culture AAV transduced CD22BBz CAR-T cells with NALM6 at 1:1 E:T ratio in fresh media supplemented with brefeldin A and 2 ng/mL IL-2.
- After 5 hours of incubation, collect and stain for surface CAR.
- Fix and permeabilize cells by fixation/permeabilization solution (BD) and add anti-IFNγ-APC or anti-TNF--FITC for intracellular staining.
- After 30 mins, wash stained cells by BD Perm/Wash™ buffer and measure cells by flow cytometry.