CAR T cells produced in vivo to treat cardiac injury
Summary
Fibrosis affects millions of people with cardiac disease and contributes to heart failure. While adoptive transfer of CAR T cells targeting fibroblast activation protein (FAP) has shown promise in reducing cardiac fibrosis, conventional CAR T cells persist for months to years and could cause chronic off-target toxicity by continuously attacking fibroblasts throughout the body, impairing wound healing.[reference:0][reference:1] A method to. ### LNP Characterization & In Vitro CAR Expression | Parameter | Result | |---------------|------------| | LNP hydrodynamic diameter | ~80 nm[reference:49] | | Polydispersity index | 0.02-0.06[reference:50] | | mRNA.
Keywords
Component: Nucleic Acid Payload; Description: Nucleoside-modified mRNA (incorporating N1-methylpseudouridine, m1Ψ) encoding FAPCAR — a chimeric antigen receptor targeting fibroblast activation protein (FAP)[reference:5]
Component: CAR Design; Description: • scFv from mouse-specific FAP monoclonal antibody (clone 73.3)[reference:6]<br>• Mouse CD3ζ and CD28 cytoplasmic signaling domains[reference:7]<br>• P2A self-cleaving peptide linked to RIAD peptide (conferring resistance to adenosine- and prostaglandin E2-mediated suppression)[reference:8]<br>• Codon-optimized for mammalian expression[reference:9]
Component: mRNA Production; Description: • In vitro transcription using T7 polymerase (MEGAScript T7 kit)[reference:10]<br>• Co-transcriptional capping with CleanCap cap1 analog[reference:11]<br>• N1-methylpseudouridine (m1Ψ) substituted for uridine[reference:12]<br>• 101-nucleotide poly(A) tail[reference:13]<br>• Cellulose purification to remove dsRNA contaminants[reference:14]
Component: Lipid Nanoparticle (LNP); Description: Proprietary formulation from Acuitas Therapeutics[reference:15]; ionizable cationic lipid, phosphatidylcholine, cholesterol, PEG-lipid[reference:16]
Component: Targeting Strategy; Description: CD5 antibody conjugation via SATA-maleimide chemistry[reference:17]:<br>• Anti-mouse CD5 (clone 53-7.3) for mouse studies[reference:18]<br>• DSPE-PEG-mal post-insertion into LNP[reference:19]<br>• Antibody functionalized with SATA to introduce sulfhydryl groups[reference:20]
Component: LNP Characterization; Description: • Size: ~80 nm hydrodynamic diameter[reference:21]<br>• Polydispersity index: 0.02-0.06[reference:22]<br>• Encapsulation efficiency: ~95% (Ribogreen assay)[reference:23]
Component: Target Antigen; Description: Fibroblast activation protein (FAP) — expressed on activated cardiac fibroblasts in injured tissue[reference:24]
Component: Injury Model; Description: Angiotensin II (1.5 μg/g/day) + phenylephrine (50 μg/g/day) via 28-day osmotic mini-pump (Alzet model 2004)[reference:25]
Parameter: Animal Model; Details: • C57BL/6N male mice[reference:26]<br>• Ai6 reporter mice (Rosa<sup>CAG-LSL-ZsGreen</sup>) from Jackson Laboratory[reference:27]<br>• Female mice excluded because they do not develop severe fibrosis in the AngII/PE injury model[reference:28]
Parameter: Injury Induction; Details: 7 days of continuous AngII/PE infusion via osmotic mini-pumps; sham controls received saline[reference:29]
Parameter: Treatment; Details: Single intravenous injection (retro-orbital) of CD5/LNP-FAPCAR or control LNPs at day 7 post-injury[reference:30]
Parameter: Echocardiography; Details: 2 weeks post-LNP administration; Fujifilm VisualSonics Ultrasound System with MS400 transducer; lightly anesthetized with Avertin[reference:31]; blinded analysis
Parameter: Sample Size; Details: • In vivo cohorts: n=7-12 biologically independent mice per condition, spread over 3-5 cohorts[reference:32]<br>• One small cohort was excluded (no positive control; excessive injury obscuring echocardiographic quantification)[reference:33]
Parameter: Control Groups; Details: • Saline-treated (sham injury)<br>• AngII/PE-injured + control T cells (MigR1)[reference:34]<br>• AngII/PE-injured + saline injection (no LNP)<br>• AngII/PE-injured + CD5/LNP without FAPCAR mRNA
Parameter: Adoptive Transfer Controls; Details: Virally engineered FAPCAR-GFP T cells (retroviral transduction) for comparison; 10⁷ cells injected[reference:35]
Analysis Category: LNP Characterization; Methods: Dynamic light scattering (Zetasizer Nano ZS) — size and PDI[reference:36]; Ribogreen assay — encapsulation efficiency[reference:37]
Analysis Category: In Vitro CAR Expression; Methods: Flow cytometry: His-tagged recombinant FAP + anti-His-PE antibody[reference:38]; T cells isolated from mouse spleens via negative selection[reference:39]
Analysis Category: In Vivo Cell Tracking; Methods: • Bioluminescence imaging (IVIS Spectrum) after luciferin injection[reference:40]<br>• Immunohistochemistry: CD3 (Abcam Ab16669), FAP (Abcam Ab207178), IgG F(ab')₂ (Jackson ImmunoResearch)[reference:41]
Analysis Category: Cardiac Function; Methods: Echocardiography (VisualSonics): fractional shortening (systolic function); trans-mitral inflow and tissue Doppler (diastolic function)[reference:42]
Analysis Category: Fibrosis Quantification; Methods: Picrosirius red staining; color deconvolution in ImageScope; at least 4 cardiac sections per mouse[reference:43]
Analysis Category: Flow Cytometry; Methods: Accuri C6 Plus or BD LSR II; antibodies: CD3, CD4, CD8, CD19, CD25, CD44, CD62L, NK1.1[reference:44]; gating: lymphocytes (FSC-A/SSC-A) → single cells (FSC-A/FSC-H)[reference:45]
Analysis Category: Trogocytosis Assay; Methods: In vitro: HEK293T cells transfected with murine FAP + luciferase; co-culture with FAPCAR T cells; luciferase loss indicates target elimination[reference:46]
Analysis Category: Histology; Methods: H&E; Picrosirius red; immunohistochemistry with DAPI counterstain; DMi8S widefield or SP8 confocal microscopy[reference:47]
Analysis Category: Statistics; Methods: Student's t-test (two conditions); one-way ANOVA with Tukey's post-hoc test (multiple conditions); R (version 4.0.5) with ggplot2[reference:48]
Parameter: LNP hydrodynamic diameter; Result: ~80 nm[reference:49]
Parameter: Polydispersity index; Result: 0.02-0.06[reference:50]
Parameter: mRNA encapsulation efficiency; Result: ~95%[reference:51]
Parameter: In vitro T cell transfection; Result: 5 μg LNP per 1 million T cells produced FAPCAR expression (flow cytometry)[reference:52]
Parameter: Fractional shortening (%); AngII/PE + Saline: Impaired; AngII/PE + CD5/LNP-FAPCAR: Significant improvement; Significance: p < 0.05[reference:53]
Parameter: Heart weight/body weight ratio; AngII/PE + Saline: Increased; AngII/PE + CD5/LNP-FAPCAR: Partially resolved; Significance: See Fig. S5A[reference:54]
Parameter: Ventricular fibrosis (%); Result: Significantly reduced only in CD5/LNP-FAPCAR treated animals[reference:55]
Parameter: Fibrosis pattern; Result: Perivascular fibrosis remained without interstitial fibrosis in treated animals[reference:56]
Parameter: CAR T cell persistence; Finding: Transient — in vivo-produced mRNA CAR T cells are short-lived[reference:57]
Parameter: Trogocytosis; Finding: FAPCAR T cells exhibited trogocytosis, retaining target antigen (FAP) as they accumulated in the spleen[reference:58]
Parameter: T cell infiltration; Finding: CD3⁺ T cells adjacent to activated fibroblasts (FAP⁺) observed 48 hours post-injection[reference:59]
Parameter: FAPCAR detection; Finding: CD3⁺ T cells in injured myocardium stained positive for FAPCAR variable fragment F(ab')₂[reference:60]
Parameter: Systemic toxicity; Result: No organ damage detected by H&E staining of heart, liver, spleen, lung, kidney[reference:61]
Parameter: Body weight; Result: No decrease after CD5/LNP-FAPCAR injection[reference:62]
Parameter: Splenocyte dynamics; Result: Count matrix provided in Table S1 (24 hours and 7 days post-injection)[reference:63]
1. Mouse model limitations: The AngII/PE injury model induces pressure overload and fibrosis but does not fully recapitulate all aspects of human heart failure. Female mice were excluded because they do not develop severe fibrosis in this model, limiting generalizability.[reference:70]
2. Small cohort exclusion: One cohort was excluded from the final data because no positive control group was included and half of the animals had significant left ventricular posterior wall abnormalities that obfuscated echocardiographic quantification.[reference:71]
3. Transient CAR T cell persistence: While transient nature is an advantage for safety, it may limit therapeutic durability; the optimal balance between efficacy duration and safety was not fully established.[reference:72]
4. No direct comparison to conventional CAR T cells: The study does not include a head-to-head comparison with adoptively transferred FAPCAR T cells (retrovirally engineered) in the same in vivo efficacy experiments, though previous work established this approach.[reference:73]
5. CD5 targeting specificity: CD5 is expressed on all T cells but also on some B cell subsets and chronic lymphocytic leukemia cells.[reference:74] Potential off-target effects on B cells were not extensively characterized.
6. Proprietary LNP formulation: The LNP used is proprietary to Acuitas Therapeutics, potentially limiting widespread adoption and manufacturing accessibility.[reference:75]
7. Potential immunogenicity: While nucleoside-modified mRNA reduces immunogenicity, repeated administration could potentially elicit anti-mRNA or anti-LNP immune responses; this was not investigated.
8. Long-term safety data: The study followed mice for up to 12 weeks for trogocytosis experiments but did not comprehensively assess long-term (≥6 months) safety or potential for delayed toxicity.[reference:76]
9. Translational gap: The study uses mouse-specific antibodies (anti-mouse CD5) and mouse FAP CAR; human translation would require humanized targeting antibodies and CAR designs.
10. No disease-modifying endpoint beyond fibrosis: While fibrosis reduction and functional improvement were demonstrated, the study did not assess long-term survival benefit, heart failure hospitalization surrogates, or effects on disease progression.
11. Conflicts of interest: Multiple authors are scientific founders and hold equity in Capstan Therapeutics (a company developing in vivo CAR T cell platforms); LNP is proprietary to Acuitas Therapeutics.[reference:77]
Report prepared based on the Science publication and supplementary materials. For full experimental details, supplementary figures, and complete references, please refer to the original article and accompanying supplemental information.
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