Chimeric antigen receptor T (CAR-T) cells, created by gene editing systems along with recombinant adeno-associated virus (rAAV), provide a promising strategy for treating leukemia. rAAVs serve as a safe and effective donor template for homology-directed repair because they can avoid integrating into the host genome. However, only a few AAV serotypes can efficiently transduce human primary T cells at low multiplicities of infection (MOIs) with high packaging efficiency. To address this problem, variants derived from an AAV2 peptide library were screened in Jurkat cells and later validated in primary T cells. A high-ranking sequence identified outside the VR-VIII region, NNSKLTV, was discovered after three rounds of selection and was named Tot3. Tot3 demonstrated transduction efficiency similar to AAV2, but at a 27-fold lower MOI. In addition, Tot3 exhibited greater packaging efficiency and reduced thermal stability. Simultaneously, programmed cell death protein 1 (PD-1) knockout and CAR overexpression were achieved in human primary T cells using Tot3, with knockout and knock-in efficiencies reaching up to 70% and 55%, respectively. These CAR-T cells demonstrated significantly enhanced antitumor activity and increased survival times in a mouse model of diffuse B cell lymphoma.
MaudeSL, LaetschTW, BuechnerJ, et al.Tisagenlecleucel in children and young adults with B-Cell lymphoblastic leukemia. N Engl J Med, 2018; 378(5):439–448; doi: 10.1056/NEJMoa1709866
2.
NarulaG, KeerthivasagamS, JainH, et al.Novel humanized CD19-CAR-T (Now talicabtagene autoleucel, Tali-cel) cells in relapsed/refractory pediatric B-acute lymphoblastic leukemia- an open-label single-arm phase-I/Ib study. Blood Cancer J, 2025; 15(1):75; doi: 10.1038/s41408-025-01279-9
3.
ZhangJ, HuY, YangJ, et al.Non-viral, specifically targeted CAR-T cells achieve high safety and efficacy in B-NHL. Nature, 2022; 609(7926):369–374; doi: 10.1038/s41586-022-05140-y
4.
CappellKMSR, YangJC, GoffSL, et al.Long-Term follow-up of anti-CD19 chimeric antigen receptor T-Cell therapy. J Clin Oncol, 2020; 38(32):3805–3815; doi: 10.1200/JCO.20
5.
ZhangC, WangXQ, ZhangRL, et al.Donor-derived CD19 CAR-T cell therapy of relapse of CD19-positive B-ALL post allotransplant. Leukemia, 2021; 35(6):1563–1570; doi: 10.1038/s41375-020-01056-6
6.
MajznerRG, MackallCL. Tumor antigen escape from CAR T-cell therapy. Cancer Discov, 2018; 8(10):1219–1226; doi: 10.1158/2159-8290.CD-18-0442
7.
WeiSC, DuffyCR, AllisonJP. Fundamental mechanisms of immune checkpoint blockade therapy. Cancer Discov, 2018; 8(9):1069–1086; doi: 10.1158/2159-8290.CD-18-0367
8.
LeeYH, LeeHJ, KimHC, et al.PD-1 and TIGIT downregulation distinctly affect the effector and early memory phenotypes of CD19-targeting CAR T cells. Mol Ther, 2022; 30(2):579–592; doi: 10.1016/j.ymthe.2021.10.004
9.
ChongEA, MelenhorstJJ, LaceySF, et al.PD-1 blockade modulates chimeric antigen receptor (CAR)-modified T cells: Refueling the CAR. Blood, 2017; 129(8):1039–1041; doi: 10.1182/blood-2016-09-738245
10.
RafiqS, YekuO, JacksonH, et al.Targeted delivery of a PD-1-blocking scFv by CAR-T cells enhances anti-tumor efficacy in vivo. Nat Biotechnol, 2018; 36(9):847–856; doi: 10.1038/nbt.4195848
11.
CherkasskyL, MorelloA, Villena-VargasJ, et al.Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition. J Clin Invest, 2016; 126(8):3130–3144; doi: 10.1172/JCI83092
12.
ZhouJE, YuJ, WangY, et al.ShRNA-mediated silencing of PD-1 augments the efficacy of chimeric antigen receptor T cells on subcutaneous prostate and leukemia xenograft. Biomed Pharmacother, 2021; 137:111339; doi: 10.1016/j.biopha.2021.111339
13.
OuyangW, JinSW, XuN, et al.PD-1 downregulation enhances CAR-T cell antitumor efficiency by preserving a cell memory phenotype and reducing exhaustion. J Immunother Cancer, 2024; 12(4):e008429; doi: 10.1136/jitc-2023-008429
14.
DuB, QinJ, LinB, et al.CAR-T therapy in solid tumors. Cancer Cell, 2025; 43(4):665–679; doi: 10.1016/j.ccell.2025.03.019
15.
MarcucciKT, JadlowskyJK, HwangWT, et al.Retroviral and lentiviral safety analysis of gene-modified T cell products and infused HIV and oncology patients. Mol Ther, 2018; 26(1):269–279; doi: 10.1016/j.ymthe.2017.10.012
16.
JadlowskyJK, HexnerEO, MarshallA, et al.Long-term safety of lentiviral or gammaretroviral gene-modified T cell therapies. Nat Med, 2025; 31(4):1134–1144; doi: 10.1038/s41591-024-03478-6
17.
AnJ, ZhangCP, QiuHY, et al.Enhancement of the viability of T cells electroporated with DNA via osmotic dampening of the DNA-sensing cGAS-STING pathway. Nat Biomed Eng, 2024; 8(2):149–164; doi: 10.1038/s41551-023-01073-7
18.
DaiX, ParkJJ, DuY, et al.One-step generation of modular CAR-T cells with AAV-Cpf1. Nat Methods, 2019; 16(3):247–254; doi: 10.1038/s41592-019-0329-7
19.
WangJ, DeClercqJJ, HaywardSB, et al.Highly efficient homology-driven genome editing in human T cells by combining zinc-finger nuclease mRNA and AAV6 donor delivery. Nucleic Acids Res, 2016; 44(3):e30; doi: 10.1093/nar/gkv1121
20.
MartinRM, IkedaK, CromerMK, et al.Highly efficient and marker-free genome editing of human pluripotent stem cells by CRISPR-Cas9 RNP and AAV6 donor-mediated homologous recombination. Cell Stem Cell, 2019; 24(5):821–828e5; doi: 10.1016/j.stem.2019.04.001
21.
EyquemJ, Mansilla-SotoJ, GiavridisT, et al.Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature, 2017; 543(7643):113–117; doi: 10.1038/nature21405
22.
BakRO, DeverDP, ReinischA, et al.Multiplexed genetic engineering of human hematopoietic stem and progenitor cells using CRISPR/Cas9 and AAV6. Elife, 2017; 6:e27873; doi: 10.7554/eLife.27873
23.
NawazW, HuangB, XuS, et al.AAV-mediated in vivo CAR gene therapy for targeting human T-cell leukemia. Blood Cancer J, 2021; 11(6):119; doi: 10.1038/s41408-021-00508-1
24.
NybergWA, ArkJ, ToA, et al.An evolved AAV variant enables efficient genetic engineering of murine T cells. Cell, 2023; 186(2):446–460e19; doi: 10.1016/j.cell.2022.12.022
25.
ZhangY, LiH, MinY-L, et al.Enhanced CRISPR-Cas9 correction of Duchenne muscular dystrophy in mice by a self-complementary AAV delivery system. Sci Adv, 2020; 6(8):Article eaay6812; doi: 10.1126/sciadv.aay6812
26.
MoyoB, BrownLBC, KhondakerII, et al.Engineering adeno-associated viral vectors for CRISPR/Cas based in vivo therapeutic genome editing. Biomaterials, 2025; 321:123314; doi: 10.1016/j.biomaterials.2025.123314
27.
ReissUM, DavidoffAM, TuddenhamEGD, et al.Sustained clinical benefit of AAV gene therapy in severe hemophilia B. N Engl J Med, 2025; 392(22):2226–2234; doi: 10.1056/NEJMoa2414783
28.
MendellJR, Al-ZaidyS, ShellR, et al.Single-Dose gene-replacement therapy for spinal muscular atrophy. N Engl J Med, 2017; 377(18):1713–1722; doi: 10.1056/NEJMoa1706198
29.
AmM. Safety and efficacy of gene transfer for Leber’s congenital Amaurosis. N Engl J Med, 2008; 358(21):2240–2248; doi: 10.1056/NEJMoa0802315
30.
WangD, TaiPWL, GaoG. Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov, 2019; 18(5):358–378; doi: 10.1038/s41573-019-0012-9
31.
WesthausA, Barba-SarasuaE, ChenY, et al.Tailoring capsid-directed evolution technology for improved AAV-mediated CAR-T generation. Mol Ther, 2025; 33(6):2801–2818; doi: 10.1016/j.ymthe.2024.12.012
32.
VineyL, BurckstummerT, EddingtonC, et al.Adeno-associated Virus (AAV) capsid chimeras with enhanced infectivity reveal a core element in the AAV genome critical for both cell transduction and capsid assembly. J Virol, 2021; 95(7):e02023–e02020; doi: 10.1128/JVI.02023-20
33.
ZhengZ, YeJ, LengM, et al.Enhanced sensitivity of neutralizing antibody detection for different AAV serotypes using HeLa cells with overexpressed AAVR. Front Pharmacol, 2023; 14:1188290; doi: 10.3389/fphar.2023.1188290
34.
DongB, NakaiH, XiaoW. Characterization of genome integrity for oversized recombinant AAV vector. Mol Ther, 2010; 18(1):87–92; doi: 10.1038/mt.2009.258
35.
OpieSR, WarringtonKHJr, Agbandje-McKennaM, et al.Identification of amino acid residues in the capsid proteins of adeno-associated virus type 2 that contribute to Heparan sulfate proteoglycan binding. J Virol, 2003; 77(12):6995–7006; doi: 10.1128/jvi.77.12.6995-7006.2003
36.
RodeL, BärC, GrossS, et al.AAV capsid engineering identified two novel variants with improved in vivo tropism for cardiomyocytes. Mol Ther, 2022; 30(12):3601–3618; doi: 10.1016/j.ymthe.2022.07.003
37.
RossiA, DupatyL, AillotL, et al.Vector uncoating limits adeno-associated viral vector-mediated transduction of human dendritic cells and vector immunogenicity. Sci Rep, 2019; 9(1):3631; doi: 10.1038/s41598-019-40071-1
38.
RiyadJM, WeberT. Intracellular trafficking of adeno-associated virus (AAV) vectors: Challenges and future directions. Gene Ther, 2021; 28(12):683–696; doi: 10.1038/s41434-021-00243-z
39.
Lopez-GordoE, ChamberlainK, RiyadJM, et al.Natural adeno-associated virus serotypes and engineered adeno-associated virus capsid variants: Tropism differences and mechanistic insights. Viruses, 2024; 16(3):442; doi: 10.3390/v16030442
40.
FerrariFK, SamulskiT, ShenkT, et al.Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors. J Virol, 1996; 70(5):3227–3234; doi: 10.1128/JVI.70.5.3227-3234
BoulchM, CazauxM, CuffelA, et al.Tumor-intrinsic sensitivity to the pro-apoptotic effects of IFN-gamma is a major determinant of CD4(+) CAR T-cell antitumor activity. Nat Cancer, 2023; 4(7):968–983; doi: 10.1038/s43018-023-00570-7
43.
GalliE, BellesiS, PansiniI, et al.The CD4/CD8 ratio of infused CD19-CAR-T is a prognostic factor for efficacy and toxicity. Br J Haematol, 2023; 203(4):564–570; doi: 10.1111/bjh.19117
44.
WangD, ZhouQ, QiuX, et al.Optimizing rAAV6 transduction of primary T cells for the generation of anti-CD19 AAV-CAR-T cells. Biomed Pharmacother, 2022; 150:113027; doi: 10.1016/j.biopha.2022.113027
45.
Lo PrestiV, CornelAM, PlantingaM, et al.Efficient lentiviral transduction method to gene modify cord blood CD8(+) T cells for cancer therapy applications. Mol Ther Methods Clin Dev, 2021; 21:357–368; doi: 10.1016/j.omtm.2021.03.015
46.
KimDY, LeeJM, MoonSB, et al.Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus. Nat Biotechnol, 2022; 40(1):94–102; doi: 10.1038/s41587-021-01009-z
47.
SheK, LiuY, ZhaoQ, et al.Dual-AAV split prime editor corrects the mutation and phenotype in mice with inherited retinal degeneration. Signal Transduct Target Ther, 2023; 8(1):57; doi: 10.1038/s41392-022-01234-1
48.
LiuY, SperlingAS, SmithEL, et al.Optimizing the manufacturing and antitumour response of CAR T therapy. Nat Rev Bioeng, 2023; 1(4):271–285; doi: 10.1038/s44222-023-00031-x
