Genetic modification of organs via ex vivo machine perfusion

Abstract

Provided herein are genetically modified organs and / or tissues comprising an immunosensor transgene, methods of generating thereof by combining genetic engineering methods with ex vivo machine perfusion, and methods of use thereof by transplantation. In some embodiments, it includes improving postoperative outcomes by genetically modifying donor organs to express diagnostic markers for rejection or infection and / or therapeutic constructs for improving graft survival.

Description

[0001]Attorney Docket No. 29539-0832WO1 / MGH 2024-313 GENETIC MODIFICATION OF ORGANS VIA EX VIVO MACHINE PERFUSION CLAIM OF PRIORITY This application claims the benefit of U.S. Provisional Application Serial No. 63 / 645,255, filed on May 10, 2024. The entire contents of the foregoing are incorporated herein by reference. FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant No. EB028782 awarded by the National Institutes of Health and Grant No. EEC1941543 awarded by the National Science Foundation. The Government has certain rights in the invention. TECHNICAL FIELD Provided herein are genetically modified organs and / or tissues comprising an immunosensor transgene, methods of generating thereof by combining genetic engineering methods with ex vivo machine perfusion, and methods of use thereof by transplantation. BACKGROUND Yearly, over 40,000 organ transplants are performed in the US alone1. The transplantation field faces critical challenges. Major examples include the limited preservation time for transplant organs; the need for lifelong systemic immunosuppression in organ transplant recipients; and the fact that invasive, late diagnostics lead to loss of transplanted organs and significant patient comorbidities and mortality (see, e.g., Piao et al., J Biomed Opt. 2018 Aug;23(8):1-14; Lepoittevin et al., Int J Mol Sci. 2022 Apr 30;23(9):4989; Henkel et al., Front Transplant. 2023 Aug 21;2:1160752). SUMMARY Gene therapy is emerging as a transformative treatment strategy, offering potential cures for a diverse range of diseases by correcting underlying genetic Attorney Docket No. 29539-0832WO1 / MGH 2024-313 defects78. Genetic modification of transplantable organs represents a promising avenue for the present challenges in the transplantation field—opening new frontiers for enhancing organ transplantation management and addressing the complexities of organ rejection.15,16Further, the penetration of warm and oxygenated perfusion technology into operating rooms worldwide offers an exciting opportunity to further enhance transplantation outcomes17. Machine perfusion maintains viable organs ex vivo for extended periods of time, minimizing ischemia-reperfusion injury (IRI), while providing precise control over physiological parameters18. It also facilitates functional assessment between organ procurement and transplantation, and provides valuable time for preconditioning and modifying organs as desired19. Leveraging machine perfusion as a platform for genetic engineering of whole organs offers the potential for targeted transduction of organs with minimal systemic immunogenicity and off-target effects, while improving the robustness of transplant organs and increasing their long-term survivability. The present disclosure provides genetically modified donor organs and / or tissues for transplantation and methods of generating thereof by combining genetic engineering methods with ex vivo machine perfusion (EVMP). Using EVMP, postoperative outcomes can be improved by genetically modifying donor organs to express diagnostic markers for rejection or infection and / or therapeutic constructs for improving graft survival. The present disclosure introduces an innovative diagnostic platform capable of detecting rejection before structural histological or visual changes manifest in the transplant organ. The diagnostic platform disclosed herein can further provide a therapeutic agent to alleviate the potential of organ rejection. Herein, key parameters were identified for genetically modifying whole organs, particularly solid organs (e.g., liver) and vascularized composite allografts (VCAs) while using EVMP. Provided herein are compositions and methods for the use of gene therapy and synthetic biology in the management of transplanted organ and / or graft outcomes (FIG. 8). Provided herein are methods of making a genetically modified organ. Methods of making a genetically modified organ provided by the present disclosure comprise ex vivo perfusion of a donor organ with an effective amount of a viral vector for transduction of an immunosensor transgene into a genome of at least one Attorney Docket No. 29539-0832WO1 / MGH 2024-313 cell of the donor organ. In some embodiments, the viral vector comprises a nucleic acid sequence encoding the immunosensor transgene, a promoter for expression of the immunosensor transgene, and a response element that directs expression of the immunosensor transgene in response to a physiological stimulus, optionally wherein the physiological stimulus is inflammation. In some embodiments, the immunosensor transgene expresses a secretable biomarker. In some embodiments, the secretable biomarker is Gaussia Luciferase (GLuc). In some embodiments, the response element is from nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). In some embodiments, the viral vector is selected from the group consisting of retroviruses, adenoviruses, oncoretroviruses, lentiviruses, spumaviruses, adeno- associated viruses, and herpes simplex viruses. In some embodiments, the viral vector is a lentivirus. In some embodiments, the viral vector comprises a nucleic sequence at least 95% identical to SEQ ID NO: 1. In some embodiments, the viral vector further comprises a nucleic acid sequence encoding at least one therapeutic protein. In some embodiments, the therapeutic protein comprises soluble GP130 (sGP130). In some embodiments, the therapeutic protein is fused to the secretable biomarker. In some embodiments, the genetically modified organ is a solid organ. In some embodiments, the solid organ is selected from the group consisting of kidney, liver, heart, lung, pancreas, and intestine. In some embodiments, the genetically modified organ is a vascularized composite allograft. In some embodiments, the vascularized composite allograft is selected from the group consisting of face, limb, long bones soft tissues, uterus, bladder, abdominal wall, musculoskeletal composite graft segments, penis, adrenal glands and thymus glands. Additionally provided herein are methods of making a genetically modified organ, the method comprising: (a) harvesting an organ from a donor; (b) placing the organ in a reservoir of a perfusion machine; (c) submerging the organ / tissue with a perfusion solution; (d) injecting the viral vector comprising a nucleic acid sequence encoding a secretable biomarker, a promoter for expression of the secretable biomarker, and a response element that directs expression of the secretable biomarker; (e) circulating the perfusate comprising the viral vector in the reservoir of the perfusion machine via closed circuit circulation; and (f) flushing the perfusate comprising the viral vector out of the organ. In some embodiments, the organ is Attorney Docket No. 29539-0832WO1 / MGH 2024-313 submerged in the perfusion solution for at least about 30 minutes before injecting the viral vector. In some embodiments, steps (c)-(f) of the method can occur at about 37°C. In some embodiments, the perfusate comprising the viral vector is circulated in the reservoir of the perfusion machine for about 5 to 12 hours. Provided herein are also methods of monitoring post-transplantation surgical outcome in a subject who has had an organ transplant. Methods of the present disclosure comprise: transplanting a genetically modified as disclosed herein organ into the subject; collecting a biological sample (e.g., a blood sample) from the subject after transplantation of the genetically modified organ; and measuring the amount of secretable biomarker in the biological sample. In some embodiments, the methods further comprise administering at least one immunosuppressant treatment to the subject to prevent or delay the onset of organ rejection if the secretable biomarker is detected in the biological sample (e.g., the blood sample). In some embodiments, the secretable biomarker is Gaussia Luciferase (GLuc). In some embodiments, GLuc is measured in the biological sample (e.g., the blood sample) by a method selected from the group consisting of a GLuc substrate assay, ELISA, Western blot, and qRT-PCR. Provided herein are also genetically modified organs comprising at least one immunosensor transgene. In some embodiments, the genetically modified organs disclosed herein comprise an immunosensor transgene wherein the immunosensor transgene expresses a secretable biomarker in response to a physiological stimulus. In some embodiments, the secretable biomarker is Gaussia Luciferase (GLuc). In some embodiments, the immunosensor transgene further expresses a therapeutic protein in response to a physiological stimulus. In some embodiments, the therapeutic protein comprises soluble GP130 (sGP130). In some embodiments, the physiological stimulus is inflammation. In some embodiments, a genetically modified organ comprising at least one immunosensor transgene is transduced with a viral vector of the present disclosure. In some embodiments, a genetically modified organ comprising at least one immunosensor transgene is generated according to any of the methods of the present disclosure. Provided herein are viral vectors comprising a nucleic acid sequence encoding an immunosensor transgene, a promoter for expression of the immunosensor transgene, and a response element that directs expression of the immunosensor Attorney Docket No. 29539-0832WO1 / MGH 2024-313 transgene in response to a physiological stimulus. In some embodiments the viral vector comprises a nucleic sequence at least 95% identical to SEQ ID NO: 1. Also contemplated herein are compositions comprising the viral vectors of the present disclosure (e.g., nanoparticles, micelles, composites, and the like). Also provided herein are kits for use in the methods disclosed herein. In some embodiments, the kits can comprise a viral vector as disclosed herein and / or one or more assay components and / or instructions for measuring a secretable biomarker in a biological sample. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. As used herein, unless otherwise specified, the term “about” means plus or minus 10%. Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims. DESCRIPTION OF DRAWINGS FIGS. 1A-1E: In vitro validation of lentiviral genetic construct showed time- and dose-dependent gene expression. (A) Schematic illustration of constitutive lentiviral construct used to engineer cells and organs throughout this study. (B) HepG2 cells were treated with increasing viral MOIs and transduction efficiency was calculated based on RFP+ cells in each group, showing a clear dose-dependent response (n = 3). Cell viability was assessed via CellTiter-Blue assay 72 h post- transduction, exhibiting an inversely proportional relationship to viral dose. (C) Representative images are shown of transduced HepG2 cells constitutively expressing RFP at increasing viral doses. (D) Engineered HEK293T cells were seeded at increasing densities and supernatant was collected over different accumulation times Attorney Docket No. 29539-0832WO1 / MGH 2024-313 for GLuc assessment, which showed clear time- and dose-dependent secretion of the biomarker. (E) Representative images are shown of (i) engineered HEK293T cells, constitutively expressing RFP, and (ii) non-engineered HEK293T cells, which showed no RFP expression. Error bars are shown as mean with range. FIGS. 2A-2E: Perfusion parameters were comparable in viral and control groups. (A) Perfusion setup consisting of: 1. Digital Peristaltic Pump 2. Oxygenation chamber 3. Bubble trap 4. Perfusion basin 5. Pressure monitor. The image of the liver shows how the organ was connected to the perfusion system and the bile collection tube. Basin was drained of perfusate for imaging purposes. Perfusion timeline with respect to viral exposure and removal, as well as perfusate addition and replacement. (B) Potassium remained stable, (C) lactate was cleared and (D) oxygen consumption remained stable throughout perfusion until end of study was reached. (E) Bile production showed a similar trend in both groups (n = 3, each), tapering off towards the end of perfusion. Error bars are shown as mean with range. Two-way ANOVA showed no significant differences between groups. TV = total volume. FIGS. 3A-3D: Ex vivo imaging showed successful viral transduction post- perfusion. (A) Bioluminescent imaging showed signal intensity increase in the (right) experimental group compared to (left) control. (B) Luminescence (photons / s) were significantly higher in the experimental (viral, 72 h, n = 3) group versus control (n = 3) (unpaired T-test, p < 0.0001). For reference, the luminescence of the 48 h viral liver (n = 1) is shown and demonstrated a signal comparable to the control group. (C) Fluorescence imaging showed hotspots that were evenly distributed throughout the liver. (D) While fluorescence signal intensity was higher in the viral group, the difference was not as pronounced and did not reach significance (unpaired T test, p = 0.4253). Error bars are shown as mean with range. ****p < 0.0001. FIGS. 4A-4D: Tissue and perfusate analyses of viral RNA and GLuc showed successful transduction. (A) Perfusate analysis for viral RNA showed successful elimination of viral particles after 24 h of exposure was reached. (B) Tissue GLuc analysis of all liver lobes grouped per replicate (normalized against total protein) showed significant levels of GLuc in the livers exposed to viral particles that reached 72 h (n = 3) (1-way ANOVA, p < 0.0001). Conversely, the liver exposed to viral particles that did not reach 72 h but 48 h due to technical failure (n = 1) showed no Attorney Docket No. 29539-0832WO1 / MGH 2024-313 significant levels compared to controls (n = 3) (1-way ANOVA, p = 0.9909). (C) Accordingly, lobe per lobe examination showed that significant GLuc levels were reached at 72 h (2-way ANOVA, column factor p < 0.0001, row factor ns). (D) Liver image shows which regions were selected for analysis. Error bars are shown as mean with range. ****p < 0.0001. FIGS. 5A-5F: Histological analysis showed comparable results between groups. Cross-section, light microscopy, x4, Hematoxylin and Eosin (H&E) staining (A, D), Periodic acid-Schiff (PAS) staining (B, E), Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (C, F). A–C shows viral tissue slides, and D–F shows control tissue slides. FIGS. 6A-6E: After 6 h of perfusion, no evidence of transduction wasdetected. (A) GLuc in the perfusate of the 6 h whole liver perfusion (viral dose: 5x108IU; n=1). (B) Vascular resistance, (C) lactate, and (D) potassium showed normal perfusion parameters. (E) Fluorescent imaging of the liver perfused with viral particles and a control liver showed that no increased fluorescence was detected, showing no evidence of transduction. FIG. 7: GLuc transgene expression was detectable in the 72 h perfusion group. Tissue analysis of GLuc mRNA via RT-qPCR revealed transgene expression in the experimental group, while this was not found in the 48 h liver (technical failure replicate) nor the control group. FIG. 8: Genetically modified organs with diagnostic capabilities can enable early detection of transplant rejection. Transplant organs can be genetically engineered to feature diagnostic sensors, enabling faster detection of rejection compared to traditional invasive biopsies. This innovation would allow for prompt intervention in clinic, ultimately enhancing patient outcomes and reducing the risk of complications and graft failure. FIGS. 9A-9F: Genetic modification of VCAs showed long-term transgene expression in plasma and tissues. (A) Experimental design describing the study timeline, starting with organ procurement, followed by machine perfusion with viral exposure and subsequent transplantation. Transgene expression was measured in plasma and tissues during the postoperative period. (B) Viral RNA measured in the perfusate during machine perfusion showed presence of viral RNA after injection and Attorney Docket No. 29539-0832WO1 / MGH 2024-313 successful wash-out after the closed circuit was opened, prior to transplantation. (C) In vivo bioluminescence imaging confirmed luminescence localized to the transplanted viral VCA. (D) Luminescence quantification showed an approximately 10-fold increase of total luminescence between groups (p=0.0176, unpaired T test) with little variation between replicates (n=3 per group). (E) Plasma GLuc levels of EF1a animals were first detected at POD 3; these levels then declined until POD 14, stabilizing after that and remained consistent for over 300 days. (F) RT-qPCR revealed significantly higher relative GLuc mRNA expression levels in the vasculature as compared to muscle and skin VCA tissues. Beta-actin (Actb) was measured as an endogenous reference gene and fold-change was calculated relative to non-viral control group levels. Statistical analysis: One-way ANOVA (* p ≤ 0.0332; ** p ≤ 0.0021; *** p ≤ 0.0002; **** p ≤ 0.0001). FIGS. 10A-10D: Temporal profile of endothelial cell viral uptake. (A) Primary rat cells showed a time-dependent response to viral treatment, with significantly higher transgene expression observed in endothelial cells compared to muscle (myocytes) and skin (fibroblasts and keratinocytes) primary cells (MOI=200; n=3). (B) Viral uptake rate in endothelial cells peaked approximately 8 h after viral treatment. Human umbilical vein endothelial cells (HUVECs) were exposed to viral vectors for varying durations of time. Control groups comprised wells with no cells but exposed to the same viral concentration (MOI=200). Supernatant samples were collected at different time points and analyzed for viral concentration using a p24 ELISA (n=4). (C) RFP fluorescence confirmed successful transduction of HUVECs 72 h post viral treatment. Remarkably, RFP+cells were detectable even with just 30 min of viral exposure. (D) Corrected total fluorescence was determined for each group by quantification of RFP+cells via ImageJ. Highest intensities were observed in cells exposed to viral vectors for 24h. Two-way ANOVA (* p ≤ 0.0332; ** p ≤ 0.0021; *** p ≤ 0.0002; **** p ≤ 0.0001). FIGS. 11A-11G: Inflammation-triggered biosensor successfully responded to rejection in vitro. (A) Schematic of genetic construct featuring inducible, inflammation-triggered promoter (NF-kB) driving the expression of two transgenes, GLuc and RFP. (B) Engineered cells containing the inducible promoter were stimulated with 20 ng / mL TNF-α for 24 h, after which the stimulation was removed. Attorney Docket No. 29539-0832WO1 / MGH 2024-313 Supernatant samples were collected hourly to assess the time-course of GLuc secretion (n=3). (C) Similarly, engineered cells were stimulated with TNF-a for 4 h, 8 h, or 24 h. The AUC was calculated for each stimulation duration and compared accordingly (n=3 each). (D) Activation of engineered cells following treatment with POD 7 rejection plasma was confirmed through RFP fluorescence imaging (left). In contrast, non-inducible cells expressing the constitutive EF1α promoter exhibited constant RFP expression regardless of plasma treatment (right). (E) GLuc secretion by cells treated with rejection plasma confirmed activation of the inducible promoter after 24 h of exposure. (F) GLuc (i) and TNF-a (ii) plasma secretion data were analyzed using a linear mixed effects model with the type of transplant (2 levels: rejection, non-rejection). Multiple comparisons were performed using Tukey’s correction under the assumption of one family for the entire transplant type. A binary classification system was then applied, and the corresponding ROC curves were generated for each pairing. The AUC was calculated for each ROC curve, showing the superior performance of GLuc as a rejection biomarker in this scenario. (G) Engineered cells were treated with increasing doses of CsA (1x = 5 μg / mL; 2x = 10 μg / mL) for 24 h. Cells were then stimulated with 20 ng / mL TNF-a for another 24 h, when the supernatant was collected and assessed for GLuc secretion. Negative control group did not receive CsA or TNF-a stimulation. Positive control group received only TNF-a stimulation after 24 h. Results indicated that the biosensor was responsive to immunosuppression, even at lower doses (n=3). Two-way ANOVA (* p ≤ 0.05; ** p ≤ 0.001; *** p ≤ 0.0001). FIGS. 12A-12J: Smart grafts outperformed other biomarkers by sensitively detecting rejection in vivo at earlier stages. (A) In vivo bioluminescence imaging showed significantly higher total luminescence in the rejected hindlimb on POD 4. (B) Plasma GLuc levels in rejection animals were significantly elevated at PODs 3 and 5 compared to the non-rejection, and rejection under IS group, suggesting early detection of rejection via blood-based biomarker secretion (n=6). (C) GLuc secretion, blinded clinical rejection score (Banff score), and selected cytokine secretion levels were analyzed using a linear mixed effects model with the type of transplant (2 levels: rejection, non-rejection). PODs (0-11) were treated as random variables for the analysis. Multiple comparisons were performed using Tukey’s correction under the Attorney Docket No. 29539-0832WO1 / MGH 2024-313 assumption of one family for the entire transplant type. A binary classification system was then applied, and the corresponding ROC curves were generated for each pairing. The AUC was calculated for each ROC curve (several cytokine AUCs were combined and the mean with range was represented for this variable), suggesting a superior performance of GLuc as a rejection biomarker. Detailed ROC curves are shown for (D) GLuc; (E) Banff score; (F) TNF-a; (G) IL-1a; (H) IL-1b and (I) VEGF. (J) Time- to-diagnose curves were generated for each variable using Kaplan-Meier analysis. Again, the status at serial time of multiple cytokines were combined to generate the curve for this variable. Results suggested that GLuc exhibited the highest probability of diagnosing rejection at earlier time points compared to other variables. One-way or Two-way ANOVA (* p ≤ 0.0332; **** p ≤ 0.0001). FIGS. 13A-13C: In vivo and ex vivo viral injections did not show signs of transduction. (A) Schematic of genetic construct featuring constitutive promoter (EF1a) driving the expression of two transgenes, GLuc and RFP. (B) The right hindlimbs of animals (i) were injected with 6 x107IU or with sterile saline, half of which was injected i.m. and half s.q. (n=1 per group). Post-injection (ii), GLuc levels in the blood did not show relevant differences between groups. (C) In a partial hindlimb transplant model, limbs were injected with 5 x 108IU, half of which was injected i.m. and half i.v. One of the limbs was flushed with saline and transplanted after 1 h WIT, and one of the limbs was subjected to 6 h SCS to extend the dwell time of the virus, before flushing and transplanting the limb. Both limbs failed by POD 4, showing extensive internal hemorrhage and even necrosis. FIGS. 14A-14E: Parameter-finding machine perfusion protocol: characterization and optimization. (A) Machine perfusion setup consisting of: 1. Digital Peristaltic Pump 2. Oxygenation chamber 3. Bubble trap 4. Perfusion basin 5. Pressure monitor. (B) Green fluorescence dye (fluorescein sodium 10%, diluted in injectable water, pH 8.3-9.8) was injected through the femoral artery after 6 h of NMP (n=1). Images were taken under a Wood lamp after dissection to reveal deeper structures, respectively. (1) Limb after dissection, showing the anterior muscle groups, (2) skin, (3) calf muscles, (4) thigh muscles, (5) fat pad, demonstrating homogenous distribution after 6 h of NMP. (C) Several conditions were tested to determine the optimal control group for comparison with the viral perfusion group. Attorney Docket No. 29539-0832WO1 / MGH 2024-313 Conditions that were tested were injected into the machine perfusion circuit in the same fashion as the viral injections: transduction enhancer (protamine sulfate in this case) diluted in PBS (n=3), protamine sulfate only (n=2) and no additives (n=8). This data was compared with viral perfusions to which PS was added (n=5). In the control groups, the addition of transduction enhancer resulted in significant edema, particularly when diluted in PBS. Therefore, the no-additive group was identified as the optimal control group. (D) Primary rat fibroblasts exhibited dose-dependent transgene expression when treated with increasing viral MOIs; cell viability was negatively affected by high viral concentrations. Supernatant GLuc levels and cell viability were determined 72 hours post-transduction; control groups consisted of primary cells that did not receive viral treatment (n=3). (E) Transduction efficiency was influenced by the assay temperature, which also impacted the viability of primary rat fibroblasts. Transduction efficiency was measured based on the percentage of RFP+cells observed 72 hours after transduction (MOI = 200; n=3). One-Way ANOVA (* p ≤ 0.0332; ** p ≤ 0.0021; *** p ≤ 0.0002). FIGs. 15A-15E: Proof-of-concept perfusions with constitutive genetic construct exhibited clear signs of transduction in vivo. (A) Perfusion parameters showed similar results between groups (n=3, each) in terms of potassium and lactate levels, glucose uptake, oxygen consumption and weight change. In the viral group, resistance was higher at the last timepoint, likely as a result of the extended perfusion time (12 h). Glucose uptake declined over time in both groups and reached stable levels, suggesting the organ was recovering from the warm ischemic time during the first 2 h, before stabilizing. The increase in potassium and lactate levels between 2 h and 11 h was due to the closed loop phase, a steady decline in both parameters was seen after opening the circuit and perfusing fresh perfusate. (B) Clinical evolution showed no clear difference between groups. However, both groups showed superficial necrosis in some of the replicates, again, most likely due to the extended perfusion time of 12 h. (C) Tissue analysis of GLuc mRNA via RT-qPCR revealed that the vasculature exhibited the highest levels of transduction in the VCA tissues, and no significant off-target effects were found in other organs. (D) Detailed analysis of the skin flap showed the topographical distribution of the GLuc signal. (E) Cross-section, light microscopy, Hematoxylin and Eosin (H&E) staining, scale bar is 100 μm. Attorney Docket No. 29539-0832WO1 / MGH 2024-313 Blinded histopathological assessment showed variable viability between replicates, with some replicates showing necrosis of skin tissue, evidenced by loss of epidermis, presence of apoptotic bodies and cell infiltration, and ischemic changes of muscle tissue evidenced by myocyte damage and size variation, interstitial edema and inflammation, suggesting compromised viability due to extended perfusion duration. Two-way ANOVA (* p ≤ 0.0332; ** p ≤ 0.0021; *** p ≤ 0.0002). FIGS. 16A-16D: Six (6) h machine perfusion protocol enabled optimal VCA viability. (A) Perfusion parameters showed similar results between groups (n=5 viral group, n=3 control group) in terms of resistance, potassium and lactate levels, glucose uptake, oxygen consumption, and weight change. Within one hour, vascular resistance decreased and remained at stable levels throughout perfusion in both groups. The increase in potassium and lactate levels between 2 h and 5 h was due to the closed loop phase, a decline in both parameters was seen after opening the circuit and perfusing fresh perfusate. In all parameters, no significant difference was found between groups at all timepoints, and no significant differences were found between 1 h and 6 h, suggesting maintained graft health. Importantly, weight change between the start and end of perfusion was <10% in both groups. (B) Out of the five animals that were transplanted with genetically modified VCAs, two were monitored for over 300 days. Representative clinical images illustrate the sustained health of the grafts and skin function, as evidenced by hair growth. Atrophy was seen beyond POD 30 in the animals that did not have the nerves reconnected. (C) Cross-section, light microscopy, Hematoxylin and Eosin (H&E) staining, scale bar is 100 μm. Histopathological assessment showed normal skin architecture without signs of injury during the postoperative period. Muscle tissue showed limited signs of edema and infiltration in the early postoperative period, and later signs of atrophy, until nerve function was regained in the replicates that underwent nerve reconnection. (D) Total luminescence of the grafts in vivo showed an approximately 4-fold increase between the viral and control limb. The same replicate was imaged again at POD 28, and displayed almost identical luminescence compared to POD 7. FIGS. 17A-17D: Rejection model for in vitro stimulation study. (A) Illustration of heterotopic VCA rejection transplant model (Brown-Norway to Lewis) and cardiac puncture at the end of study (POD 7) for blood collection. Engineered Attorney Docket No. 29539-0832WO1 / MGH 2024-313 cells were generated featuring the inducible promoter NF-kB and stimulated with plasma from rejection animals (n=3). (B) Clinical evolution of VCA transplants showed a normal flap with hair growth in the non-rejection group and signs of severe rejection in the rejection group at POD 7. (C) Cross-section, light microscopy, Hematoxylin and Eosin (H&E) staining, scale bar is 100 μm. Histopathological assessment showed (left) normal skin architecture in the non-rejection group and (right) frank necrosis evidenced by complete loss of the epidermis and dermal architecture, cell infiltration and presence of apoptotic bodies. (D) Plasma cytokine and chemokine panel compared levels in rejection, non-rejection and healthy animal groups. Signal was normalized to POD 1 background. Two-way ANOVA (* p ≤ 0.0332; ** p ≤ 0.0021; *** p ≤ 0.0002; **** p ≤ 0.0001). FIGS. 18A-18F: Genetically engineered VCAs exhibited signs of rejection postoperatively in the mismatch group. (A) Perfusion parameters showed similar results between groups (n=6, each) for all perfusion parameters throughout 6 h of NMP. Glucose consumption showed a significant difference between both groups at 5 h, but this was not seen in any of the other time points. The increase in potassium and lactate levels between 1 h and 5 h was due to the closed loop phase, a steady decline in both parameters was seen after opening the circuit and perfusing fresh perfusate at 5 h 45 m. (B) Representative clinical pictures showed erythema on POD 5 and lack of hair growth on POD 11 in the rejection group. Upon palpation the rejection group showed indurated muscle tissue and edema. (C) RT-qPCR revealed significantly higher relative GLuc mRNA expression levels in VCA tissues compared to off-target non-VCA control tissues. Beta-actin (Actb) was measured as an endogenous reference gene and fold-change was calculated relative to non-rejection control group. (D) Cross-section, light microscopy, Hematoxylin and Eosin (H&E) staining, scale bar is 100 μm. (E) Blinded histopathological assessment showed that rejection was observed on POD 9 in the rejection group and increased in severity over time. Slides showed cell-mediated rejection as evidenced by cellular infiltration resulting in a mean Banff score of 1 on POD 9, and a mean score of 3.3 on POD 11. (F) Selected plasma cytokine panel (i. TNF-a; ii. IL-1a; iii. IL-1b; iv. IL-6; v. VEGF; vi. MCP-1) revealed significantly higher levels of pro-inflammatory cytokines and chemokines in Attorney Docket No. 29539-0832WO1 / MGH 2024-313 rejection group. Statistical analysis: One-way or Two-way ANOVA (* p ≤ 0.0332; ** p ≤ 0.0021; *** p ≤ 0.0002; **** p ≤ 0.0001). FIGS. 19A-19I: Histological analysis of off-target organs showed no relevant pathological changes. Cross-section, light microscopy, Hematoxylin and Eosin (H&E) staining, error bar is 100 μm. Blinded histopathological assessment of the (A) heart, (B) lung, (C) liver, (D) spleen, (E) kidney, (F) intestine (G) skin, (H) muscle and (I) femoral vessels of the contralateral hindlimb with the non-rejection group shown on the left and rejection group shown on the right, showed no relevant pathological changes in any of the off-target organs. FIG. 20: Vector map of inflammation-responsive genetic construct, pLV-NF- kB-GLuc-IRES-mRFP. FIG. 21: Vector map of the pLV-EF1a-hIL6ST[ORF024283]-GLuc-IRES- mRFP plasmid. FIG. 22: Blinded clinical rejection score (Banff score) in GLuc / sGP130 VCA tissues compared to off-target non-VCA control and rejection VCAs tissues. FIG. 23: Clinical evolution showed erythema in rejection VCAs from around POD 5 onwards whereas the onset of erythema was delayed in GLuc / sGP130 VCA tissues. DETAILED DESCRIPTION Local monitoring and delivery of immunosuppression is critical to making allografts safer and more accessible. The inadequacy of current organ rejection diagnostic methods can compromise both graft survival and patient health. Traditional diagnostics rely on unspecific clinical signs, general trends in blood work, and invasive biopsies, often leading to substantial patient discomfort, potential complications and delayed detection and thereby treatment of rejection4,40,41. Moreover, current methods may yield false-positive or false-negative results due to significant changes that can occur in the patient's body postoperatively, such as infections, diet, unrelated medications, and in the case of biopsies, inhomogeneity of the rejection process in the transplanted organ, which can obscure rejection indicators42. To address the need for more accessible, non-invasive, and precise diagnostic tools, the present disclosure provides a platform for genetically engineering whole organs that integrates diagnostic features directly into the graft, (e.g., solid Attorney Docket No. 29539-0832WO1 / MGH 2024-313 organs (e.g., liver) and VCAs). By engineering whole organs, the present disclosure provides a novel method for real-time rejection monitoring through systemic detection of a secretable biomarker. Provided herein are essential transduction parameters, including time-, dose- and tissue type-dependence, and showed that the vasculature exhibited the highest transduction levels. This was particularly advantageous considering the important role of endothelial cells in IRI and, especially, rejection43-46. These process parameters and cell targets of transduction can inform the process and doses considered at large animal and human scale. Allometric scaling has begun to be explored in gene therapy, evaluating if in vitro kinetic data can be used to inform in vivo model- dose justification47,48. By applying appropriate scaling factors derived from robust mathematical models, in vitro viral uptake rates, with endothelial cells as a first approximation for organ uptake, can be utilized to predict the viral dose needed in vivo to achieve optimal transduction efficiencies. Interestingly, other groups have used similar or equal doses of viral particles in both small and large animal models to achieve whole organ transduction21,49, which is likely to result in the overtreatment of smaller organs and lead to issues such as toxicity and immunogenicity. Simulating genetic expression that fits experimental data in ex vivo organ perfusion using allometric scaling techniques, as performed herein, provides better confidence in maximizing effectiveness of gene modification and minimizing risk. Moreover, as larger organs likely require significantly higher viral doses, other concerns arise around the logistics of viral vector production, especially in good manufacturing practice settings. A key innovation of methods and compositions disclosed herein lies in their early detection capabilities. Remarkably, the secretable biomarkers as shown herein were able to detect rejection approximately six days earlier than conventional histopathological assessment. This early activation of NF-kB was likely attributed to its crucial role in innate immunity51, linked to the initial stages of rejection37,52, as well as the specificity to local inflammatory events, which was facilitated by the targeted viral transduction approach disclosed herein. Cytokine-based rejection monitoring, while effective in specific contexts, lacks the spatial and temporal specificity that engineered organs can provide53,54. Cytokines, like other endogenous Attorney Docket No. 29539-0832WO1 / MGH 2024-313 biomarkers, can fluctuate due to a variety of factors unrelated to rejection and are influenced at a systemic multiorgan level, which makes them unreliable biomarkers for continuous monitoring. An important consideration, however, is that NF-kB is a ubiquitous inflammatory transcription factor and could also be activated by non- rejection inflammatory events, potentially leading to false positive results. Surprisingly, NFkB, the inflammation-based promoter drove the secretion of GLuc in a partial rejection model, but not in non-rejection model. As such, the constructs of the present disclosure respond to rejection, not to nonspecific surgical inflammation, thus providing a valuable biomarker specific for graft failure. This specificity suggests the existence of an activation threshold for the promoter, increasing confidence that false-positive signals are unlikely to occur. Previous efforts in the field of transplant diagnostics have largely focused on implantable bioelectronic systems. Although these methods show promise, they still present some disadvantages. Madhvapathy et al. developed a microfabricated temperature biosensor that continuously monitors the local temperature of the transplanted organ via thermal conductivity42. While this approach offers valuable insights, it requires the implantation of a device and relies solely on temperature as an indicator of early acute rejection, which can be affected by various extraneous factors, such as metabolic changes, physiological variations and other environmental factors, potentially limiting its accuracy. Other studies have investigated the use of a sensor- integrated flexible patch that employs electrical impedance spectroscopy to detect rejection in heart transplants61; a subcutaneous microporous scaffold that functions as an immune cell activation surveillance method62; a granzyme B-responsive fluorescent probe that can monitor the secretion pattern of CD8+cytotoxic T cells63; and even a granzyme B-responsive nanosensor that can non-invasively detect rejection via a colorimetric readout from the urine64. Notably, many of these recent approaches rely on adaptive immunity as the primary indicator of tissue rejection. In contrast, methods provided by the present disclosure can detect the early activation of innate immune responses, allowing for identification of rejection at a local level much sooner in the process. This can be crucial from a clinical standpoint, as it enables more effective treatment management and timely interventions. Engraftment of genetically engineered cell sensors into transplantable organs using ex vivo machine Attorney Docket No. 29539-0832WO1 / MGH 2024-313 perfusion has been demonstrated16,65. However, a key limitation of this approach, is the short half-life of these cells and their natural clearance from the body. This challenge substantially restricts their use as sensors for long-term monitoring of organ rejection. Methods of the present disclosure make use of lentiviral vectors as gene delivery tools for stable and long-term transduction of genes of interest66(e.g., an immunosensor transgene). Lentiviruses have gained popularity in gene therapy applications due to their ability to integrate into non-dividing cells with a relatively safe integration profile, especially when using 3rdor 4thgeneration plasmids which avoid risk of generation of a replication-competent virus67. This makes them excellent candidates for genetically engineering whole organs, where the majority of the tissues consist of non-dividing cells. Their unique properties enable more effective and sustained expression of immunosensor and / or therapeutic protein genes, as demonstrated herein, enhancing the potential for successful organ engineering and long-term monitoring. Furthermore, the machine perfusion protocol disclosed herein includes a critical final step where viral vectors are flushed-out, mitigating concerns about the immunogenicity associated with their presence in vivo. Importantly, significant immune responses against the viral vectors of the present disclosure were not observed in the treated groups, nor was transduction in off-target tissues detected. This demonstrates that the methods provided herein are both localized and safe, ensuring targeted action without unintended side effects. Ex vivo genetic modification of grafts using machine perfusion presents a promising approach to improve graft function and modulate immune responses while minimizing risks of off-target effects and systemic immunogenicity in vivo. As used in the methods herein, normothermic machine perfusion provides a unique platform that allows for genetically modification of whole organs and / or tissues ex vivo. These methods of the present disclosure offer isolated access to organs to expose them to high vector dosages, with the possibilities of preserving them in an environment optimal for both organ preservation and gene therapy, as well as metabolic and genomic monitoring for quality control. The gene therapies provided herein can integrate secretable biomarkers (e.g., GLuc) into transplantable organs and / or tissues Attorney Docket No. 29539-0832WO1 / MGH 2024-313 for real-time monitoring and early rejection detection, reducing the need for invasive biopsies and / or therapeutic proteins to prevent graft rejection. Genetic Constructs Described herein are methods and compositions for use with donor organs that dynamically self-monitor for rejection following transplant and / or deliver therapeutic proteins by genetically engineering said donor organs with viral vectors encoding genetic constructs (FIG. 8). The genetically modified organs (e.g., donor organs) provided herein sense their environments through a variety of cell signaling mechanisms, and respond to changing conditions by altering gene expression. These genetically modified organs (also referred to as “smart grafts”) of the present disclosure not only perform their required functions but also possess enhanced resilience, reduced immunogenicity, and / or the capability to provide real-time health monitoring. The term “donor,” as used herein, refers to a subject from whom an organ is obtained by surgery, wherein that organ is referred to as a “donor organ.” Donor organs of the present disclosure are subjected to genetic modification according to the methods disclosed herein prior to being transplanted into a recipient. The term “recipient,” as used herein, refers to a subject to whom an organ is transferred by surgery. A donor and / or a recipient of the present disclosure can include a mammal e.g., a human, a non-human primate, a rodent (e.g., mouse, rat, rabbit), an ungulate (e.g., ovine, bovine, equine, caprine, porcine species), a canine, and a feline. As used herein a donor organ can be a solid organ. In some embodiments, a solid organ can be kidney, liver, heart, lung, pancreas, and / or intestine. A donor organ for use herein can also be a vascularized composite allograft (VCA). According to the National Organ Transplant Act (42 CFR 121.2), a VCA is a body part that is vascularized and requires blood flow by surgical connection of blood vessels to function after transplantation; contains multiple tissue types; recovered from a donor as an anatomical / structural unit; and transplanted into a recipient as an anatomical / structural unit. In some embodiments, a VCA can be a face, limb, long bones soft tissues, uterus, bladder, abdominal wall, musculoskeletal composite graft segments, penis, and glands (e.g., vascularized glands such as adrenal glands and thymus glands.). Attorney Docket No. 29539-0832WO1 / MGH 2024-313 Genetically modified organs of the present disclosure respond to changing conditions by inducing transgene expression via the activation of proteins known as transcription factors. Transcription factor mediated gene activation is a central paradigm of genetic regulation. Transcription factors bind to discrete regions of DNA known as response elements within the regulatory regions of genes. The present disclosure provides genetic constructs that drive protein biomarker expression (e.g., secretable biomarker) and / or therapeutic protein expression in response to molecular and physiological cues of inflammation. A donor organ is generally subject to reperfusion injury that occurs with restoration of blood during organ implantation in the recipient, a condition known as ischemia reperfusion injury (IRI). The resulting IRI leads to graft inflammation and localized activation of the innate immune system (see, e.g., Mori et al., Immunol Rev. 2014 Mar;258(1):132-44). The genetic constructs disclosed herein provide for insertion of an immunosensor transgene into a donor organ, thus generating a genetically modified organ, as provided by the present disclosure. An immunosensor generally refers to a biosensor that combines a biological recognition mechanism with a transducer, which generates a measurable signal (e.g., a secretable biomarker) in response to changes in the concentration of a given biomolecule. Here, the genetic constructs disclosed herein comprise a response element (e.g., an inflammatory response element) to generate expression of the immunosensor transgene – a measurable protein biomarker (e.g., a secretable biomarker) – in response to localized activation of the innate immune system at the site of transplant. Currently, localized inflammation of the graft can only be accurately determined by biopsy of the transplanted organ. By secreting a measurable blood-based biomarker (e.g., GLuc) in response to inflammation, the genetically modified organs, which comprise an immunosensor transgene as provided herein, essentially self-monitor for rejection. In some embodiments, a genetic construct disclosed herein comprises at least one response element that directs expression of the secretable biomarker (e.g., expression of the immunosensor transgene) in response to a physiological stimulus, e.g., inflammation. In some embodiments, the response element is an inflammatory response element. In some embodiments, the response element can be NF-κB, interferon-sensitive response element (ISRE), heat-shock consensus element (HSE), Attorney Docket No. 29539-0832WO1 / MGH 2024-313 activating protein-1 (AP-1), signal transducer and activator of transcription 1 (STAT1), signal transducer and activator of transcription 3 (STAT3), signal transducer and activator of transcription 5 (STAT5), signal transducer and activator of transcription 6 (STAT6), Interferon Regulatory Factor 4 (IRF4), Interferon Regulatory Factor 3 (IRF3), Interferon Regulatory Factor 9 (IRF9), activating transcription factor-2 (ATF2), C / EBPα (CCAAT / enhancer-binding protein alpha), C / EBPβ (CCAAT / enhancer-binding protein beta), Smad2 and Smad3 (Smad2 / 3), Smad4 and Smad5 (Smad4 / 5), or any combination thereof. In some embodiments, the response element is from nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). In some embodiments, the NF-κB inflammatory response element is an isolated nucleic acid comprising the sequence: GGGAATTTCCCGGGAATTTCCGGGACTTTCCGGGAATTTCCCGGGAATTTCC GGGACTTTCC (SEQ ID NO: 2). In some embodiments, a genetic construct disclosed herein comprises at least one NF-κB response element. In some embodiments, a genetic construct disclosed herein comprises at least two NF-κB response elements. In some embodiments, a genetic construct disclosed herein comprises a strong minimal promoter. In some embodiments, a strong minimal promoter suitable for use herein can be a thymidine kinase (TK) minimal promoter, a minimal CMV, a SV40 minimal promoter, a EF1α core promoter, or any combination thereof. In some embodiments, a genetic construct disclosed herein comprises a thymidine kinase (TK) minimal promoter. In some embodiments, a TK minimal promoter can contribute to the genetic construct’s high sensitivity. In some embodiments, the full promoter comprises two NF-κB response elements upstream of a TK minimal promoter. In some embodiments, a genetic construct disclosed herein comprises at least one a sequence encoding a secretable biomarker. In some embodiments, a secretable biomarker disclosed herein is a secreted protein selected from an enzyme, a peptide hormone, or a peptide or protein antigen. Non-limiting examples of secretable biomarkers can include alkaline phosphatase, a human chorionic gonadotropin, a human carcinoembryonic antigen (CEA), colon cancer secreted protein 2, Cathepsin B, Gaussia luciferase (Gluc), and Metridia luciferase (MLuc). In some embodiments, a secretable biomarker can be a secreted luciferase protein. In some embodiments, a Attorney Docket No. 29539-0832WO1 / MGH 2024-313 secreted luciferase protein can be Gaussia Luciferase (GLuc). In some embodiments, the GLuc secretable biomarker is an isolated nucleic acid comprising the sequence: ATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCA AGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTT CGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTG CCGCTGGAGGTGCTCAAAGAGATGGAAGCCAATGCCCGGAAAGCTGGCTGCA CCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAA GAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCA CAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCA AGGACTTGGAGCCCATGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGA CTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTG CTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGG GCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAA (SEQ ID NO: 3). In some embodiments, a genetic construct of the present disclosure comprises the structure as provided in FIG. 20, pLV-NF-kB-GLuc-IRES-mRFP. In some embodiments, a genetic construct of the present disclosure (e.g., pLV-NF-kB-GLuc- IRES-mRFP) is an isolated nucleic acid comprising the sequence: 1 AATGTAGTCT TATGCAATAC TCTTGTAGTC TTGCAACATG GTAACGATGA 51 GTTAGCAACA TGCCTTACAA GGAGAGAAAA AGCACCGTGC ATGCCGATTG 101 GTGGAAGTAA GGTGGTACGA TCGTGCCTTA TTAGGAAGGC AACAGACGGG 151 TCTGACATGG ATTGGACGAA CCACTGAATT GCCGCATTGC AGAGATATTG 201 TATTTAAGTG CCTAGCTCGA TACATAAACG GGTCTCTCTG GTTAGACCAG 251 ATCTGAGCCT GGGAGCTCTC TGGCTAACTA GGGAACCCAC TGCTTAAGCC 301 TCAATAAAGC TTGCCTTGAG TGCTTCAAGT AGTGTGTGCC CGTCTGTTGT 351 GTGACTCTGG TAACTAGAGA TCCCTCAGAC CCTTTTAGTC AGTGTGGAAA 401 ATCTCTAGCA GTGGCGCCCG AACAGGGACT TGAAAGCGAA AGGGAAACCA 451 GAGGAGCTCT CTCGACGCAG GACTCGGCTT GCTGAAGCGC GCACGGCAAG 501 AGGCGAGGGG CGGCGACTGG TGAGTACGCC AAAAATTTTG ACTAGCGGAG 551 GCTAGAAGGA GAGAGATGGG TGCGAGAGCG TCAGTATTAA GCGGGGGAGA 601 ATTAGATCGC GATGGGAAAA AATTCGGTTA AGGCCAGGGG GAAAGAAAAA 651 ATATAAATTA AAACATATAG TATGGGCAAG CAGGGAGCTA GAACGATTCG 701 CAGTTAATCC TGGCCTGTTA GAAACATCAG AAGGCTGTAG ACAAATACTG 751 GGACAGCTAC AACCATCCCT TCAGACAGGA TCAGAAGAAC TTAGATCATT 801 ATATAATACA GTAGCAACCC TCTATTGTGT GCATCAAAGG ATAGAGATAA 851 AAGACACCAA GGAAGCTTTA GACAAGATAG AGGAAGAGCA AAACAAAAGT 901 AAGACCACCG CACAGCAAGC GGCCGCTGAT CTTCAGACCT GGAGGAGGAG 951 ATATGAGGGA CAATTGGAGA AGTGAATTAT ATAAATATAA AGTAGTAAAA 1001 ATTGAACCAT TAGGAGTAGC ACCCACCAAG GCAAAGAGAA GAGTGGTGCA 1051 GAGAGAAAAA AGAGCAGTGG GAATAGGAGC TTTGTTCCTT GGGTTCTTGG 1101 GAGCAGCAGG AAGCACTATG GGCGCAGCGT CAATGACGCT GACGGTACAG 1151 GCCAGACAAT TATTGTCTGG TATAGTGCAG CAGCAGAACA ATTTGCTGAG 1201 GGCTATTGAG GCGCAACAGC ATCTGTTGCA ACTCACAGTC TGGGGCATCA 1251 AGCAGCTCCA GGCAAGAATC CTGGCTGTGG AAAGATACCT AAAGGATCAA 1301 CAGCTCCTGG GGATTTGGGG TTGCTCTGGA AAACTCATTT GCACCACTGC 1351 TGTGCCTTGG AATGCTAGTT GGAGTAATAA ATCTCTGGAA CAGATTTGGA 1401 ATCACACGAC CTGGATGGAG TGGGACAGAG AAATTAACAA TTACACAAGC 1451 TTAATACACT CCTTAATTGA AGAATCGCAA AACCAGCAAG AAAAGAATGA 1501 ACAAGAATTA TTGGAATTAG ATAAATGGGC AAGTTTGTGG AATTGGTTTA 1551 ACATAACAAA TTGGCTGTGG TATATAAAAT TATTCATAAT GATAGTAGGA 1601 GGCTTGGTAG GTTTAAGAAT AGTTTTTGCT GTACTTTCTA TAGTGAATAG 1651 AGTTAGGCAG GGATATTCAC CATTATCGTT TCAGACCCAC CTCCCAACCC 1701 CGAGGGGACC CGACAGGCCC GAAGGAATAG AAGAAGAAGG TGGAGAGAGA Attorney Docket No. 29539-0832WO1 / MGH 2024-313 1751 GACAGAGACA GATCCATTCG ATTAGTGAAC GGATCTCGAC GGTATCGCTA 1801 GCTTTTAAAA GAAAAGGGGG GATTGGGGGG TACAGTGCAG GGGAAAGAAT 1851 AGTAGACATA ATAGCAACAG ACATACAAAC TAAAGAATTA CAAAAACAAA 1901 TTACAAAAAT TCAAAATTTT ACTAGTATCA ACTTTGTATA GAAAAGTTGG 1951 GGAATTTCCC GGGAATTTCC GGGACTTTCC GGGAATTTCC CGGGAATTTC 2001 CGGGACTTTC CGGGAATTTC CCGGGAATTT CCGGGACTTT CCGGGAATTT 2051 CCCGGGAATT TCCGGGACTT TCCGGCGGTG TCCCCGGAAG AAATATATTT 2101 GCATGTCTTT AGTTCTATGA TGACACAAAC CCCGCCCAGC GTCTTGTCAT 2151 TGGCGAATTC GAACACGCAG ATGCAGTCGG GGCGGCCAAG TTTGTACAAA 2201 AAAGCAGGCT GCCACCATGG GAGTCAAAGT TCTGTTTGCC CTGATCTGCA 2251 TCGCTGTGGC CGAGGCCAAG CCCACCGAGA ACAACGAAGA CTTCAACATC 2301 GTGGCCGTGG CCAGCAACTT CGCGACCACG GATCTCGATG CTGACCGCGG 2351 GAAGTTGCCC GGCAAGAAGC TGCCGCTGGA GGTGCTCAAA GAGATGGAAG 2401 CCAATGCCCG GAAAGCTGGC TGCACCAGGG GCTGTCTGAT CTGCCTGTCC 2451 CACATCAAGT GCACGCCCAA GATGAAGAAG TTCATCCCAG GACGCTGCCA 2501 CACCTACGAA GGCGACAAAG AGTCCGCACA GGGCGGCATA GGCGAGGCGA 2551 TCGTCGACAT TCCTGAGATT CCTGGGTTCA AGGACTTGGA GCCCATGGAG 2601 CAGTTCATCG CACAGGTCGA TCTGTGTGTG GACTGCACAA CTGGCTGCCT 2651 CAAAGGGCTT GCCAACGTGC AGTGTTCTGA CCTGCTCAAG AAGTGGCTGC 2701 CGCAACGCTG TGCGACCTTT GCCAGCAAGA TCCAGGGCCA GGTGGACAAG 2751 ATCAAGGGGG CCGGTGGTGA CTAAACCCAG CTTTCTTGTA CAAAGTGGGC 2801 CCCTCTCCCT CCCCCCCCCC TAACGTTACT GGCCGAAGCC GCTTGGAATA 2851 AGGCCGGTGT GCGTTTGTCT ATATGTTATT TTCCACCATA TTGCCGTCTT 2901 TTGGCAATGT GAGGGCCCGG AAACCTGGCC CTGTCTTCTT GACGAGCATT 2951 CCTAGGGGTC TTTCCCCTCT CGCCAAAGGA ATGCAAGGTC TGTTGAATGT 3001 CGTGAAGGAA GCAGTTCCTC TGGAAGCTTC TTGAAGACAA ACAACGTCTG 3051 TAGCGACCCT TTGCAGGCAG CGGAACCCCC CACCTGGCGA CAGGTGCCTC 3101 TGCGGCCAAA AGCCACGTGT ATAAGATACA CCTGCAAAGG CGGCACAACC 3151 CCAGTGCCAC GTTGTGAGTT GGATAGTTGT GGAAAGAGTC AAATGGCTCT 3201 CCTCAAGCGT ATTCAACAAG GGGCTGAAGG ATGCCCAGAA GGTACCCCAT 3251 TGTATGGGAT CTGATCTGGG GCCTCGGTGC ACATGCTTTA CATGTGTTTA 3301 GTCGAGGTTA AAAAAACGTC TAGGCCCCCC GAACCACGGG GACGTGGTTT 3351 TCCTTTGAAA AACACGATGA TAATATGGCC ACAACCATGG CCTCCTCCGA 3401 GGACGTCATC AAGGAGTTCA TGCGCTTCAA GGTGCGCATG GAGGGCTCCG 3451 TGAACGGCCA CGAGTTCGAG ATCGAGGGCG AGGGCGAGGG CCGCCCCTAC 3501 GAGGGCACCC AGACCGCCAA GCTGAAGGTG ACCAAGGGCG GCCCCCTGCC 3551 CTTCGCCTGG GACATCCTGT CCCCTCAGTT CCAGTACGGC TCCAAGGCCT 3601 ACGTGAAGCA CCCCGCCGAC ATCCCCGACT ACTTGAAGCT GTCCTTCCCC 3651 GAGGGCTTCA AGTGGGAGCG CGTGATGAAC TTCGAGGACG GCGGCGTGGT 3701 GACCGTGACC CAGGACTCCT CCCTGCAGGA CGGCGAGTTC ATCTACAAGG 3751 TGAAGCTGCG CGGCACCAAC TTCCCCTCCG ACGGCCCCGT AATGCAGAAG 3801 AAGACCATGG GCTGGGAGGC CTCCACCGAG CGGATGTACC CCGAGGACGG 3851 CGCCCTGAAG GGCGAGATCA AGATGAGGCT GAAGCTGAAG GACGGCGGCC 3901 ACTACGACGC CGAGGTCAAG ACCACCTACA TGGCCAAGAA GCCCGTGCAG 3951 CTGCCCGGCG CCTACAAGAC CGACATCAAG CTGGACATCA CCTCCCACAA 4001 CGAGGACTAC ACCATCGTGG AACAGTACGA GCGCGCCGAG GGCCGCCACT 4051 CCACCGGCGC CTAACAACTT TATTATACAT AGTTGATCAA TTCCGATAAT 4101 CAACCTCTGG ATTACAAAAT TTGTGAAAGA TTGACTGGTA TTCTTAACTA 4151 TGTTGCTCCT TTTACGCTAT GTGGATACGC TGCTTTAATG CCTTTGTATC 4201 ATGCTATTGC TTCCCGTATG GCTTTCATTT TCTCCTCCTT GTATAAATCC 4251 TGGTTGCTGT CTCTTTATGA GGAGTTGTGG CCCGTTGTCA GGCAACGTGG 4301 CGTGGTGTGC ACTGTGTTTG CTGACGCAAC CCCCACTGGT TGGGGCATTG 4351 CCACCACCTG TCAGCTCCTT TCCGGGACTT TCGCTTTCCC CCTCCCTATT 4401 GCCACGGCGG AACTCATCGC CGCCTGCCTT GCCCGCTGCT GGACAGGGGC 4451 TCGGCTGTTG GGCACTGACA ATTCCGTGGT GTTGTCGGGG AAGCTGACGT 4501 CCTTTCCATG GCTGCTCGCC TGTGTTGCCA CCTGGATTCT GCGCGGGACG 4551 TCCTTCTGCT ACGTCCCTTC GGCCCTCAAT CCAGCGGACC TTCCTTCCCG 4601 CGGCCTGCTG CCGGCTCTGC GGCCTCTTCC GCGTCTTCGC CTTCGCCCTC 4651 AGACGAGTCG GATCTCCCTT TGGGCCGCCT CCCCGCATCG GGAATTCCCG Attorney Docket No. 29539-0832WO1 / MGH 2024-313 4701 CGGTTCGCTT TAAGACCAAT GACTTACAAG GCAGCTGTAG ATCTTAGCCA 4751 CTTTTTAAAA GAAAAGGGGG GACTGGAAGG GCTAATTCAC TCCCAACGAA 4801 GACAAGATCT GCTTTTTGCT TGTACTGGGT CTCTCTGGTT AGACCAGATC 4851 TGAGCCTGGG AGCTCTCTGG CTAACTAGGG AACCCACTGC TTAAGCCTCA 4901 ATAAAGCTTG CCTTGAGTGC TTCAAGTAGT GTGTGCCCGT CTGTTGTGTG 4951 ACTCTGGTAA CTAGAGATCC CTCAGACCCT TTTAGTCAGT GTGGAAAATC 5001 TCTAGCAGTA GTAGTTCATG TCATCTTATT ATTCAGTATT TATAACTTGC 5051 AAAGAAATGA ATATCAGAGA GTGAGAGGAA CTTGTTTATT GCAGCTTATA 5101 ATGGTTACAA ATAAAGCAAT AGCATCACAA ATTTCACAAA TAAAGCATTT 5151 TTTTCACTGC ATTCTAGTTG TGGTTTGTCC AAACTCATCA ATGTATCTTA 5201 TCATGTCTGG CTCTAGCTAT CCCGCCCCTA ACTCCGCCCA TCCCGCCCCT 5251 AACTCCGCCC AGTTCCGCCC ATTCTCCGCC CCATGGCTGA CTAATTTTTT 5301 TTATTTATGC AGAGGCCGAG GCCGCCTCGG CCTCTGAGCT ATTCCAGAAG 5351 TAGTGAGGAG GCTTTTTTGG AGGCCTAGGG ACGTACCCAA TTCGCCCTAT 5401 AGTGAGTCGT ATTACGCGCG CTCACTGGCC GTCGTTTTAC AACGTCGTGA 5451 CTGGGAAAAC CCTGGCGTTA CCCAACTTAA TCGCCTTGCA GCACATCCCC 5501 CTTTCGCCAG CTGGCGTAAT AGCGAAGAGG CCCGCACCGA TCGCCCTTCC 5551 CAACAGTTGC GCAGCCTGAA TGGCGAATGG GACGCGCCCT GTAGCGGCGC 5601 ATTAAGCGCG GCGGGTGTGG TGGTTACGCG CAGCGTGACC GCTACACTTG 5651 CCAGCGCCCT AGCGCCCGCT CCTTTCGCTT TCTTCCCTTC CTTTCTCGCC 5701 ACGTTCGCCG GCTTTCCCCG TCAAGCTCTA AATCGGGGGC TCCCTTTAGG 5751 GTTCCGATTT AGTGCTTTAC GGCACCTCGA CCCCAAAAAA CTTGATTAGG 5801 GTGATGGTTC ACGTAGTGGG CCATCGCCCT GATAGACGGT TTTTCGCCCT 5851 TTGACGTTGG AGTCCACGTT CTTTAATAGT GGACTCTTGT TCCAAACTGG 5901 AACAACACTC AACCCTATCT CGGTCTATTC TTTTGATTTA TAAGGGATTT 5951 TGCCGATTTC GGCCTATTGG TTAAAAAATG AGCTGATTTA ACAAAAATTT 6001 AACGCGAATT TTAACAAAAT ATTAACGCTT ACAATTTAGG TGGCACTTTT 6051 CGGGGAAATG TGCGCGGAAC CCCTATTTGT TTATTTTTCT AAATACATTC 6101 AAATATGTAT CCGCTCATGA GACAATAACC CTGATAAATG CTTCAATAAT 6151 ATTGAAAAAG GAAGAGTATG AGTATTCAAC ATTTCCGTGT CGCCCTTATT 6201 CCCTTTTTTG CGGCATTTTG CCTTCCTGTT TTTGCTCACC CAGAAACGCT 6251 GGTGAAAGTA AAAGATGCTG AAGATCAGTT GGGTGCACGA GTGGGTTACA 6301 TCGAACTGGA TCTCAACAGC GGTAAGATCC TTGAGAGTTT TCGCCCCGAA 6351 GAACGTTTTC CAATGATGAG CACTTTTAAA GTTCTGCTAT GTGGCGCGGT 6401 ATTATCCCGT ATTGACGCCG GGCAAGAGCA ACTCGGTCGC CGCATACACT 6451 ATTCTCAGAA TGACTTGGTT GAGTACTCAC CAGTCACAGA AAAGCATCTT 6501 ACGGATGGCA TGACAGTAAG AGAATTATGC AGTGCTGCCA TAACCATGAG 6551 TGATAACACT GCGGCCAACT TACTTCTGAC AACGATCGGA GGACCGAAGG 6601 AGCTAACCGC TTTTTTGCAC AACATGGGGG ATCATGTAAC TCGCCTTGAT 6651 CGTTGGGAAC CGGAGCTGAA TGAAGCCATA CCAAACGACG AGCGTGACAC 6701 CACGATGCCT GTAGCAATGG CAACAACGTT GCGCAAACTA TTAACTGGCG 6751 AACTACTTAC TCTAGCTTCC CGGCAACAAT TAATAGACTG GATGGAGGCG 6801 GATAAAGTTG CAGGACCACT TCTGCGCTCG GCCCTTCCGG CTGGCTGGTT 6851 TATTGCTGAT AAATCTGGAG CCGGTGAGCG TGGGTCTCGC GGTATCATTG 6901 CAGCACTGGG GCCAGATGGT AAGCCCTCCC GTATCGTAGT TATCTACACG 6951 ACGGGGAGTC AGGCAACTAT GGATGAACGA AATAGACAGA TCGCTGAGAT 7001 AGGTGCCTCA CTGATTAAGC ATTGGTAACT GTCAGACCAA GTTTACTCAT 7051 ATATACTTTA GATTGATTTA AAACTTCATT TTTAATTTAA AAGGATCTAG 7101 GTGAAGATCC TTTTTGATAA TCTCATGACC AAAATCCCTT AACGTGAGTT 7151 TTCGTTCCAC TGAGCGTCAG ACCCCGTAGA AAAGATCAAA GGATCTTCTT 7201 GAGATCCTTT TTTTCTGCGC GTAATCTGCT GCTTGCAAAC AAAAAAACCA 7251 CCGCTACCAG CGGTGGTTTG TTTGCCGGAT CAAGAGCTAC CAACTCTTTT 7301 TCCGAAGGTA ACTGGCTTCA GCAGAGCGCA GATACCAAAT ACTGTTCTTC 7351 TAGTGTAGCC GTAGTTAGGC CACCACTTCA AGAACTCTGT AGCACCGCCT 7401 ACATACCTCG CTCTGCTAAT CCTGTTACCA GTGGCTGCTG CCAGTGGCGA 7451 TAAGTCGTGT CTTACCGGGT TGGACTCAAG ACGATAGTTA CCGGATAAGG 7501 CGCAGCGGTC GGGCTGAACG GGGGGTTCGT GCACACAGCC CAGCTTGGAG 7551 CGAACGACCT ACACCGAACT GAGATACCTA CAGCGTGAGC TATGAGAAAG 7601 CGCCACGCTT CCCGAAGAGA GAAAGGCGGA CAGGTATCCG GTAAGCGGCA Attorney Docket No. 29539-0832WO1 / MGH 2024-313 7651 GGGTCGGAAC AGGAGAGCGC ACGAGGGAGC TTCCAGGGGG AAACGCCTGG 7701 TATCTTTATA GTCCTGTCGG GTTTCGCCAC CTCTGACTTG AGCGTCGATT 7751 TTTGTGATGC TCGTCAGGGG GGCGGAGCCT ATGGAAAAAC GCCAGCAACG 7801 CGGCCTTTTT ACGGTTCCTG GCCTTTTGCT GGCCTTTTGC TCACATGTTC 7851 TTTCCTGCGT TATCCCCTGA TTCTGTGGAT AACCGTATTA CCGCCTTTGA 7901 GTGAGCTGAT ACCGCTCGCC GCAGCCGAAC GACCGAGCGC AGCGAGTCAG 7951 TGAGCGAGGA AGCGGAAGAG CGCCCAATAC GCAAACCGCC TCTCCCCGCG 8001 CGTTGGCCGA TTCATTAATG CAGCTGGCAC GACAGGTTTC CCGACTGGAA 8051 AGCGGGCAGT GAGCGCAACG CAATTAATGT GAGTTAGCTC ACTCATTAGG 8101 CACCCCAGGC TTTACACTTT ATGCTTCCGG CTCGTATGTT GTGTGGAATT 8151 GTGAGCGGAT AACAATTTCA CACAGGAAAC AGCTATGACC ATGATTACGC 8201 CAAGCGCGCA ATTAACCCTC ACTAAAGGGA ACAAAAGCTG GAGCTGCAAG 8251 CTT (SEQ ID NO: 1). Table 1 provides the components of the genetic construct pLV-NF-kB-GLuc-IRES- mRFP. TABLE 1: Components of the Inflammation-Responsive Genetic Construct Attorney Docket No. 29539-0832WO1 / MGH 2024-313 Also provided herein are therapeutic proteins engineered for secretion from the genetically modified organ. In some embodiments, a genetic construct disclosed herein comprises at least one a response element that directs expression of a therapeutic protein in response to a physiological stimulus, e.g., inflammation. In some embodiments, a genetic construct disclosed herein comprises at least one a response element that directs expression of a secretable biomarker and a therapeutic protein in response to a physiological stimulus, e.g., inflammation. In some embodiments, the therapeutic protein can be soluble tumor necrosis factor receptor-1 (sTNFR1), soluble IL-6 receptor (sIL6R), interleukin-1 receptor antagonist (IL-1Ra), interleukin 10 (IL-10), or any combination thereof. In some embodiments, the therapeutic protein is an inhibitor of the pro-inflammatory cytokine interleukin 6 (IL- 6). In some embodiments, the therapeutic protein is soluble glycoprotein 130 (sGP130). In some embodiments, the sGP130 therapeutic protein comprising the amino acid sequence: MSAPRIWLAQALLFFLTTESIGQLLEPCGYIYPEFPVVQRGSNFTAICVLKE ACLQHYYVNASYIVWKTNHAAVPREQVTVINRTTSSVTFTDVVLPSVQLTCN ILSFGQIEQNVYGVTMLSGFPPDKPTNLTCIVNEGKNMLCQWDPGRETYLET NYTLKSEWATEKFPDCQSKHGTSCMVSYMPTYYVNIEVWVEAENALGKVSSE SINFDPVDKVKPTPPYNLSVTNSEELSSILKLSWVSSGLGGLLDLKSDIQYR TKDASTWIQVPLEDTMSPRTSFTVQDLKPFTEYVFRIRSIKDSGKGYWSDWS EEASGTTYEDRPSRPPSFWYKTNPSHGQEYRSVRLIWKALPLSEANGKILDY EVILTQSKSVSQTYTVTGTELTVNLTNDRYVASLAARNKVGKSAAAVLTIPS PHVTAAYSVVNLKAFPKDNLLWVEWTPPPKPVSKYILEWCVLSENAPCVEDW QQEDATVNRTHLRGRLLESKCYQITVTPVFATGPGGSESLKAYLKQAAPARG PTVRTKKVGKNEAVLAWDQIPVDDQNGFIRNYSISYRTSVGKEMVVHVDSSH TEYTLSSLSSDTLYMVRMAAYTDEGGKDGPEFTFTTPKFAQGEIEAIVVPVC LAFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNS KDQMYSDGNFTDVSVVEIEANNKKPCPDDLKSVDLFKKEKVSTEGHSSGIGG SSCMSSSRPSISSNEENESAQSTASTVEYSTVVHSGYRHQVPSVQVFSRSES TQPLLDSEERPEDLQLVDSVDGGDEILPRQPYFKQNCSQPEACPEISHFERS NQVLSGNEEDFVRLKQQQVSDHISQPYGSEQRRLFQEGSTADALGTGADGQM ERFESVGMETTIDEEIPKSYLPQTVRQGGYMPQ (SEQ ID NO: 4). In some embodiments, an isolated nucleic acid sequence encoding for sGP130 can be added into the genetic construct pLV-NF-kB-GLuc-IRES-mRFP disclosed herein. Using the response element on NF-KB to drive sGP130 secretion, the therapeutic biomolecule can be expressed in response to inflammatory activation. In some embodiments, the secretable biomarker can be fused to the N or C terminus ofthe therapeutic protein. In some embodiments, a single viral vector as provided herein Attorney Docket No. 29539-0832WO1 / MGH 2024-313 can express the secretable biomarker and the therapeutic proteins as two separate proteins. In some embodiments, two viral vectors as provided herein, one expressing the secretable biomarker and a second for expressing the therapeutic protein may be administered to the subject simultaneously. Nucleic acid molecules comprising expression vectors can be used, e.g., for in vitro expression of the secretable biomarker. The nucleic acids encoding the secretable biomarker can be inserted in an expression vector, to make an expression construct. A number of suitable vectors are known in the art, e.g., viral vectors, recombinant bacterial or eukaryotic plasmids. For example, the expression construct includes a response element and a coding region encoding a secretable biomarker as described herein, as well as one or more of a promoter sequence, e.g., a promoter sequence that restricts expression to a selected cell type, a conditional promoter, or a strong general promoter; another enhancer sequence; untranslated regulatory sequences, e.g., a 5’ untranslated region (UTR), a 3’ UTR; a polyadenylation site; and / or an insulator sequence. Such sequences are known in the art, and the skilled artisan would be able to select suitable sequences. See, e.g., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14; Vancura (ed.), TRANSCRIPTIONAL REGULATION: METHODS AND PROTOCOLS (Methods in Molecular Biology (Book 809)) Humana Press; 2012 edition (2011) and other standard laboratory manuals. In some embodiments, the sequence of a protein or nucleic acid used in a composition or method described herein is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to a reference sequence set forth herein. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90% or 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or Attorney Docket No. 29539-0832WO1 / MGH 2024-313 nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available on the world wide web at gcg.com), using the default parameters, e.g., a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. Viral Vectors The use of viral vectors is an approach to systemically and dynamically release secretable biomarkers (e.g., GLuc), and optionally therapeutic proteins (e.g., sGP130) in response to localized inflammation. Infection of cells within the organ / tissue with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within the viral vector, e.g., by a nucleic acid contained in the viral vector, are expressed efficiently in cells that have taken up viral vector nucleic acid. Viral vector systems used herein are also considered to be “gene therapies” and are suitable for use in genomic integration of the immunosensor transgene as described herein. Expression constructs of such components can be administered in any effective carrier, e.g., any formulation or composition capable of effectively delivering the component gene to cells in vivo. Approaches include insertion of the gene in viral vectors, including recombinant retroviruses, adenovirus, adeno- associated virus, lentivirus, and herpes simplex virus- 1, or recombinant bacterial or eukaryotic plasmids. Viral vectors transfect cells directly; plasmid DNA can be delivered naked or with the help of, for example, cationic liposomes (lipofectamine) or derivatized (e.g., antibody conjugated), polylysine conjugates, gramacidin S, Attorney Docket No. 29539-0832WO1 / MGH 2024-313 artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPCri precipitation carried out in vivo. A viral gene delivery system useful in the present methods utilizes adenovirus- derived vectors. The genome of an adenovirus can be manipulated, such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See, for example, Berkner et al., BioTechniques 6:616 (1988); Rosenfeld et al., Science 252:431-434 (1991); and Rosenfeld et al., Cell 68: 143-155 (1992). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, or Ad7 etc.) are known to those skilled in the art. Recombinant adenoviruses can be advantageous in certain circumstances, in that they are capable of infecting non dividing cells and can be used to infect a wide variety of cell types, including epithelial cells (Rosenfeld et al., (1992) supra). Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity. Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situ, where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al., supra; Haj-Ahmand and Graham, J. Virol. 57:267 (1986). Another viral vector system useful for delivery of nucleic acids is the adeno- associated virus (AAV). Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see Muzyczka et al., Curr. Topics in Micro and Immunol.158:97-129 (1992). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al., Am. J. Respir. Cell. Mol. Biol. 7:349-356 (1992); Samulski et al., J. Virol. 63:3822-3828 (1989); and McLaughlin et al., J. Virol. 62: 1963-1973 (1989). Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is Attorney Docket No. 29539-0832WO1 / MGH 2024-313 limited to about 4.5 kb. An AAV vector such as that described in Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985) can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al., Proc. Natl. Acad. Sci. USA 81 :6466-6470 (1984); Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1985); Wondisford et al., Mol. Endocrinol. 2:32-39 (1988); Tratschin et al., J. Virol. 51 :611-619 (1984); and Flotte et al., J. Biol. Chem. 268:3781-3790 (1993). Retrovirus vectors can be used as a recombinant gene delivery system for the transfer of exogenous genes in vivo, including in human cells. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host. The development of specialized cell lines (termed “packaging cells”) which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are characterized for use in gene transfer for gene therapy purposes (for a review see Miller, Blood 76:271 (1990)). A replication defective retrovirus can be packaged into infectious virions using standard techniques involving helper plasmids or packaging cell lines that supply the necessary viral proteins in trans. These virions can then be used to infect target cells for gene delivery. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Ausubel, et ah, eds., Current Protocols in Molecular Biology, Greene Publishing Associates, (1989), Sections 9.10-9.14, and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are known to those skilled in the art. Examples of suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include Y(¾r, *FCre, Y2 and YAih. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and / or in vivo (see for example Eglitis, et al. (1985) Science 230: 1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991)Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640- Attorney Docket No. 29539-0832WO1 / MGH 2024-313 7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89: 10892-10895; Hwu et al. (1993) J. Immunol.150:4104-4115; U.S. Patent No. 4,868,116; U.S. Patent No. 4,980,286; PCT Application WO 89 / 07136; PCT Application WO 89 / 02468; PCT Application WO 89 / 05345; and PCT Application WO 92 / 07573. In some embodiments, the retrovirus vector is a lentiviral vector. Lentiviral vectors (LV) are a type of retrovirus that can deliver genes into both dividing and non-dividing cells, making them valuable tools for gene therapy (see, e.g., Patel & Misra: Gene delivery using viral vectors. In: Challenges in delivery of therapeutic genomics and proteomics. Elsevier; 2011: 207–270). For delivery of the immunosensor transgenes of the present disclosure, LVs are suitable due to their ability to carry bulky and complex transgenes and sustain robust and long-term expression in a broad range of dividing and non-dividing cells in vitro and in vivo. Types of LV can include self-inactivating (SIN) lentiviral vectors, integration- deficient lentiviral vectors (IDLVs), and / or conditional expression lentiviral vectors. LV are generally known in the art and are suitable for use herein (see, e.g., Milone & O'Doherty, Leukemia. 2018 Jul;32(7):1529-1541; Nóbrega et al., (2020). Viral Vectors for Gene Therapy. In: A HANDBOOK OF GENE AND CELL THERAPY. Springer. doi.org / 10.1007 / 978-3-030-41333-0_3; Ottaviano & Qasim, Leukemia. 2025 Apr 8. doi: 10.1038 / s41375-025-02585-8). In some embodiments, an organ / tissue disclosed herein can be transduced with genetic construct-encoded lentiviral vectors (LV), retroviral vectors (RV), or adeno- associated vectors (AAV) using transduction enhancing reagents (e.g. retronectin, protamine sulfate, polybrene, vectofusin-1, Sirion AdenoBOOST™, Sirion LentiBOOST), or enhancer-free physical co-localization (e.g. centrifugation-based spinoculation by Cytiva Sepax C-pro or Miltenyi Prodigy). As disclosed herein, the amount of transduction enhancing reagents used during transduction is important for long term graft survival as high amounts of transduction enhancing reagents causes major edema in the donor organ. In some embodiments, an organ / tissue disclosed herein can be transduced with genetic construct-encoded viral vectors (e.g., AAV, LV) and low amounts (e.g., about 10 to about 20 μg / mL of perfusate; e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μg / mL of perfusate) of at least one Attorney Docket No. 29539-0832WO1 / MGH 2024-313 transduction enhancing reagent. In some embodiments, at least one transduction enhancing reagent is protamine sulfate, polybrene, or a combination thereof. Methods of transducing an organ / tissue are disclosed herein wherein the method utilizes machine perfusion. Whereas the details of machine perfusion are discussed below, the dose of the viral vector is an important factor for uptake of the genetic constructs / immunosensor transgenes disclosed herein. In some embodiments, contacting a cell or organ / tissue with a nucleic acid, a vector, or a composition as described herein includes “introducing” or “delivering” (directly or indirectly) with the nucleic acid, vector, or composition into the cell or organ / tissue by facilitating or effecting uptake or absorption into the cell, tissue, organ. Introducing a nucleic acid, a vector, or a composition as described herein into a cell or an organ / tissue can be ex vivo and / or in vivo. In some embodiments, a viral vector comprising the isolated nucleic acids (genetic constructs) disclosed herein is delivered to a donor organ. In some embodiments, a viral vector disclosed herein is delivered to a donor organ via machine perfusion. In some embodiments, a viral vector (e.g., AAV, LV) is delivered to a donor organ via machine perfusion at an amount suitable for transduction efficiency (e.g., genomic integration rate). Transduction efficiency suitable for use herein can be about 50% to about 100% of the cells within the organ demonstrating genomic integration of the nucleic acid (e.g., immunosensor transgene) after the viral vector was delivered to the organ according to the methods disclosed herein. As used herein, an organ demonstrating genomic integration of the nucleic acid (e.g., immunosensor transgene) after the viral vector was delivered to the organ according to the methods disclosed herein is referred to thereafter as a genetically modified organ. In some embodiments, a viral vector disclosed herein is an AAV and is delivered to a donor organ via machine perfusion. In some embodiments, an AAV is delivered to a donor organ via machine perfusion at an amount that is at least about 1x1010IU (infectious units). In some embodiments, an AAV is delivered to a donor organ via machine perfusion at an amount that is higher than about 1x1010IU. In some embodiments, an AAV is delivered to a donor organ via machine perfusion for about 4 or more hours. In some embodiments, an AAV is delivered to a donor organ Attorney Docket No. 29539-0832WO1 / MGH 2024-313 at an amount that is higher than about 1x1010IU via machine perfusion for about 4 or more hours. In some embodiments, a viral vector disclosed herein is a LV and is delivered to a donor organ via machine perfusion. In some embodiments, a LV is delivered to a donor organ via machine perfusion at an amount that is at least about 1x1012IU. In some embodiments, a LV is delivered to a donor organ via machine perfusion at an amount that is higher than about 1x108IU. In some embodiments, a LV is delivered to a donor organ via machine perfusion for about 5 or more hours. In some embodiments, a LV is delivered to a donor organ via machine perfusion for at least about or about 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, or more than 12 hours. In some embodiments, a LV is delivered to a donor organ at an amount thatis about 1x1012IU via machine perfusion for about 5 to 12 hours.Machine perfusion In the methods provided herein, a viral vector comprising the isolated nucleic acids (genetic constructs) disclosed herein is delivered to a donor organ via machine perfusion. A "perfusion machine," as used herein refers to a machine, system or device that comprises a pump, a reservoir to hold an organ (e.g., solid organ or VCA), a second reservoir to hold a perfusate, and tubing to connect the reservoir holding the organ and the perfusate reservoir. In some embodiments, the perfusion system further comprises tubing that connects a blood vessel or a lymphatic vessel of an organ with the perfusate reservoir. For example, a perfusion system can be a Hugo Sachs / Harvard Apparatus, Kidney Assist™ system, OrganOX system, Radnoti system, ARK Kidney system, and Aferetica PerLife® system. A "perfusate," as used herein, refers to a liquid composition that runs through a blood vessel or lymphatic vessel of an organ ex vivo or otherwise permeates an organ ex vivo. A perfusate can comprise at least one of a buffer, an inorganic salt, an amino acid, a substrate for metabolism, a hormone, a vasodilator, a tonicity agent, an oxygenation agent, an anti-oxidant, an anti-inflammatory agent, an anti-coagulant, and / or an anti-microbial agent. In some embodiments, a viral vector comprising the isolated nucleic acids (genetic constructs) disclosed herein can be included in the perfusate. In some embodiments, a transduction enhancing reagent disclosed herein Attorney Docket No. 29539-0832WO1 / MGH 2024-313 can be included in the perfusate. The amounts of viral vectors and / or transduction enhancing reagents to be included in the perfusate are described herein. In some embodiments, a viral vector comprising the isolated nucleic acids (genetic constructs) disclosed herein is delivered to a donor organ via ex vivo machine perfusion before transplanting the donor organ into the recipient. Ex vivo perfusion is also called normothermic perfusion and comprises a machine which keeps organs at body temperature by continuously pumping or perfusing blood or a bloodless solution of nutrients, proteins and oxygen, through them. Ex vivo perfusion can reduce ischemic injury time and allow for graft evaluation. As demonstrated herein, the temperature during perfusion can affect transduction efficiency. In some embodiments, normothermic machine perfusion is at a temperature ranging from about 20°C to about 40°C; about 21° C to about 39°C; about 22° C to about 38°C; or about 23°C to about 37°C. In some embodiments, normothermic machine perfusion is at least about or about 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C. In some embodiments, normothermic machine perfusion is about 37°C. Methods disclosed herein can include a loading phase. The loading phase can include subjecting the organ / tissue to normothermic machine perfusion with a normothermic perfusion, e.g., by flushing, perfusing, and / or submerging the organ / tissue with the normothermic perfusion solution at a normothermic temperature (e.g., 35-40°C, e.g., about 37°C). A viral vector comprising the isolated nucleic acids (genetic constructs) is delivered to the donor organ (e.g., solid organ, VCA) after the start of perfusion (e.g., after the loading phase). In some embodiments, a viral vector comprising the isolated nucleic acids is delivered to the donor organ at least about or about 30 minutes, 45 minutes, or 1 hour after the start of perfusion. In some embodiments, a viral vector comprising the isolated nucleic acids is delivered to the donor organ via the perfusate. In some embodiments, a viral vector comprising the isolated nucleic acids is injected into the perfusate. In some embodiments, a viral vector comprising the isolated nucleic acids is injected into the perfusate through the bubble trap. In some embodiments, a viral vector comprising the isolated nucleic acids is injected into the perfusate through an inflow port close to the arterial cannula. Attorney Docket No. 29539-0832WO1 / MGH 2024-313 In some embodiments, normothermic machine perfusion should continuously circulate a perfusate comprising the viral vector comprising the isolated nucleic acids (genetic constructs). This can be accomplished by establishing a closed circuit. In some embodiments, perfusate comprising the viral vector comprising the isolated nucleic acids circulated in the closed system for at least 5 hours. In some embodiments, perfusate comprising the viral vector comprising the isolated nucleic acids circulated in the closed system for at least about or about 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12 or more than 12 hours. In some embodiments, perfusate comprising the viral vector comprising the isolated nucleic acids circulated in the closed system for about 5 hours. In some embodiments, perfusate comprising the viral vector comprising the isolated nucleic acids circulated in the closed system for about 12 hours. Prior to removing the organ from machine perfusion and before transplantation, the viral vector should be flushed out of the organ in order to avoid systemic off-target effects after transplantation. In some embodiments, the viral vector is flushed out of the organ by perfusing the organ with fresh perfusate. This is accomplished by removing the closed system and adding a perfusate solution that does not contain the viral vector. In some embodiments, the viral vector is flushed out of the organ by perfusing the organ with fresh perfusate for at least 5 to 15 minutes. In some embodiments, the viral vector is flushed out of the organ by perfusing the organ with fresh perfusate for at least about or about 15 minutes. Methods of Use Provided herewith are organs and methods of preparing them for transplantation. In some embodiments, the organs are procured from a donor. In some embodiments, an organ is explanted from one subject (the donor), submitted to the materials and methods as described herein, and transplanted into another subject (the recipient). In some embodiments, an organ is explanted from one subject (the donor), submitted to the materials and methods as described herein, and transplanted into the same subject (the donor and the recipient are one subject). In some embodiments, the donors are human subjects. In some embodiments, the donors are non-human subjects (e.g., xenotransplantation). In some embodiments, the recipients are human subjects in need of an organ transplant. A “subject in need thereof’ as utilized herein may Attorney Docket No. 29539-0832WO1 / MGH 2024-313 refer to a subject in need of treatment for a disease or disorder for which the treatment is organ transplantation. Non-limiting examples of such diseases can include chronic kidney failure, heart failure, valvular heart disease, congenital heart disease, coronary artery disease, cardiomyopathy, cystic fibrosis, pulmonary edema, emphysema, pulmonary hypertension, Type I insulin-dependent diabetes mellitus, congenital liver defects, and short bowel syndrome. Methods herein further comprise transplanting the organ (e.g., genetically modified organ) into a subject. Organ transplantation is a medical procedure in which an organ is removed from one body and placed in the body of a recipient, to replace a damaged or missing organ. The donor and recipient may be at the same location, or organs may be transported from a donor site to another location. An allograft is a transplant of an organ or tissue between two genetically non-identical members of the same species. Due to the genetic difference between the organ and the recipient, the recipient's immune system may identify the organ as foreign and attempt to destroy it, causing transplant rejection. In addition, in cases of stem cell, bone marrow or other hematopoietic transplants the immune cells of the transplant attack the host cells. This is called Graft-versus-host disease (GvHD). “Graft” refers to transplanted, or donated tissue, and “host” refers to the tissues of the recipient. Transplantation recipients often receive prophylactic treatment to suppress the immune system after the transplant. These treatments can continue after transplantation. Immunosuppressant treatment includes, but is not limited to, calcineurin, tacrolimus, axatilimab, ruxolitinib, belumosudil, ibrutinib, corticosteroids as well as photopheresis. The methods provided herein may reduce the need for or the levels of immunosuppressant treatment needed to avoid transplant rejection by allowing for monitoring and / or treatment of post-transplantation surgical outcome(s). The genetically modified organ described herein can act as an in-situ cell- based biosensor for reporting and responding to the state of a graft. The present invention also provides a method for prevention or reducing rejection of a transplanted organ in a subject without the need for a biopsy. Methods disclosed herein can prevent or reduce rejection of a transplanted organ in a subject herein by monitoring the secreted biomarker to assess the state of the graft. In some embodiments, the methods comprise introducing the viral vector comprising the Attorney Docket No. 29539-0832WO1 / MGH 2024-313 isolated nucleic acids (genetic constructs) into a transplantable donor organ prior to transplantation into a subject. Once the genetically modified organ is transplanted, a blood sample can be collected from the recipient and subjected to the appropriate analysis for analyzing the secretable biomarker disclosed herein (e.g., GLuc). A secretable biomarker for use herein can be measured in a biological sample collected from a subject according to methods known in the art (see, e.g., Dinis-Oliveira et al., Forensic Sci Res (2017) Jan 16;1(1):42-51). In some embodiments, a biological sample for use herein can comprise whole blood, serum, plasma, and / or urine. Where the secretable biomarker is GLuc, the appropriate analysis can comprise a GLuc substrate assay, protein quantification (ELISA, Western blot), and / or mRNA quantification (qRT-PCR) in the biological sample (e.g., blood sample). In some embodiments, detection of GLuc in the biological sample (e.g., blood sample) is indicative of local inflammation at the site of the donor organ. In some embodiments, where GLuc is detected in the biological sample (e.g., blood sample), at least one immunosuppressant can be administered to the subject to prevent or delay the onset of organ rejection. In some embodiments, where GLuc is detected in the biological sample (e.g., blood sample), the amount of immunosuppressant administered to the subject can be increased to prevent or delay the onset of organ rejection. In some embodiments, transplantation of a genetically modified organ as disclosed herein can prevent or offset the time until transplant rejection. For genetically modified organs comprising an immunosensor transgene that further encodes a therapeutic protein (e.g., sGP130), the localized inflammatory stage surrounding the transplanted organ can be treated with the secreted therapeutic protein, thus preventing or offset the time until transplant rejection. EXAMPLES The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. Materials and Methods The following materials and methods were used in the Examples below. Attorney Docket No. 29539-0832WO1 / MGH 2024-313 Production of Lentiviral Vectors for In Vitro Studies Self-inactivating lentiviral vectors (LV) were generated using triple- transfection methods in adherent human embryonic kidney 293T (HEK293T) cells (see, e.g., Tan, et al., Front Bioeng Biotechnol. 9, 796991 (2021)). Briefly, HEK293T cells were expanded in Dulbecco’s modified Eagle’s / F12 medium DMEM / F-12 media (Thermo Fisher Scientific) supplemented with 10% v / v FBS and 1% v / v antibiotic-antimycotic solution (Thermo Fisher Scientific). Cells were seeded at 40% confluency the day before transfection. HEK293T cells were co-transfected with either pLV-EF1a-GLuc-IRES-mRFP or pLV-NF-kB-GLuc-IRES-mRFP plasmids, purchased and sequence-verified by vendor (VectorBuilder Inc., Chicago, IL, USA), and two packaging plasmids, psPAX2 - plasmid #12260 (Addgene, Watertown, MA, USA) and pMD2.G - plasmid #12259 (Addgene), at a molar ratio of 3:2:1. As demonstrated herein, the pLV-NF-kB-GLuc-IRES-mRFP plasmid is an inflammation- responsive genetic construct (see vector map at FIG. 20) comprising a nucleic acid sequence of SEQ ID NO: 1. Transfection reagent polyethylenimine (Polyplus, New York, NY, USA) was also used for enhanced transfection efficiencies. Transfection culture was carried out for 72 h until supernatant was collected, centrifuged and filtered through a 0.45 mm PES membrane filter. Viral solution was then purified using a sucrose-gradient protocol97. Briefly, 3 mL of 20% sucrose was added to conical bottom 30 mL ultracentrifuge tubes and, very carefully, 26 mL of viral solution was overlaid, creating two separate layers. Samples were ultracentrifuged at 26,000 rpm for 90 min at 4 °C. Pure lentiviral vectors were pellet to the bottom of the tubes. Supernatant was discarded and viral pellet (invisible) was resuspended in 3 mL of sterile PBS. Vector titer was determined via qPCR using a Lentiviral titration kit (Applied Biologic Materials, Richmond, BC, Canada) on Quant Studio 3 (Thermo Fisher Scientific). Quality control assays were carried out for determination of transduction efficiencies (TE). Increasing doses of viral particles were added to HEK293T cells along with 25 mg / mL of transduction reagent Protamine Sulfate. After 72 h, TE was determined based on GLuc secretion in the supernatant and mRFP fluorescence. Lentiviral batches with transduction efficiencies >40% were deemed acceptable for use in in vitro analysis and ex vivo perfusion studies. GLuc was Attorney Docket No. 29539-0832WO1 / MGH 2024-313 selected due to its sensitive and reliable reporting as a secreted biomarker ex vivo94as well as in vivo98. LV Production for In Vivo Studies Ultra-purified VSV-G pseudo 3rd-generation lentiviral vectors were purchased from VectorBuilder Inc. and shipped in 1 mL aliquots of >109IU / mL, all of which were purified by sucrose cushion ultracentrifugation and concentrated by ultrafiltration. Each viral batch was delivered with its respective certificate of analysis (COA) containing p24 ELISA titration results, sterility testing for bacteria and fungi, mycoplasma and endotoxin detection and transduction efficiency results. Validation of Viral Construct InVitro HepG2 cells (ATCC) were cultured in DMEM-F12 medium (Thermo Fisher) supplemented with 10% FBS (Gibco, Life Technologies) and 1% antibiotic / antifungal v / v (Thermo Fisher). 5 × 104cells were plated in 24-well plates and lentiviral vectors expressing EF1α-GLuc-IRES-RFP were added at increasing MOIs (50, 100 and 150 IU / cell), along with 25 mg / mL of transduction reagent Protamine Sulfate (Thermo Fisher). After 72 h, transduction efficiency was assessed via RFP fluorescence using Celigo Imaging Cytometer (Nexcelom Bioscience) and GLuc concentration in the supernatant. Cellular viability was assessed using CellTiter-Blue Viability Reagent (Promega) by measuring fluorescence (excitation at 579 nm, emission at 584 nm) with a Varioskan plate reader. Measurements were taken immediately after adding 20 μl of the reagent to each well (time point 0) and continued up to 240 min afterward. Additionally, cell count and viability were determined using the NucleoCounter® instrument (Chemometec). Ex Vivo Liver Perfusions Ethics approval: The experimental protocol for live vertebrate animals was approved by the Institutional Care and Use Committee (IACUC) of MGH under protocol number 2011N000111 and Biosafety protocol number 2012B000042, and all experiments were carried out in accordance with guidelines established in said protocol. Experimental design: Whole livers were procured from adult Lewis rats and underwent one of the following conditions: (1) Control: 72-h normothermic machine Attorney Docket No. 29539-0832WO1 / MGH 2024-313 perfusion (NMP) of whole liver with no viral treatment (n = 3); or (2) Viral treatment: 72-h NMP of whole liver with exposure to lentivirus containing the genetic construct outlined in Fig. 1 (n = 4). Mimicking in vitro metrics99, viral exposure lasted for 24 h and transduction assessment occurred 48 h after viral exposure (at 72 h). Perfusate preparation: Recovery perfusate was composed from a base of 500 mL Williams’ Medium E (with sodium bicarbonate, without L-glutamine, without phenol red) (Sigma-Aldrich, St. Louis, MO, USA) into which 285.7143 nM polyethylene glycol (PEG) 35000 kDa (Sigma-Aldrich, 81310), 61.1527 μM dexamethasone (water-soluble) (Sigma-Aldrich, D4902), 150.5344 μM bovine serum albumin (Sigma-Aldrich, A7906), 9.5227 mM sodium bicarbonate (Sigma-Aldrich, S6014), 4.998 mM L-glutathione (Sigma-Aldrich, G4251), 0.01 U / mL insulin (Eli Lilly and Company, Cambridge, MA, USA, 0002821501), 10 U / mL sodium heparin (Pfizer Inc, NYC, NY, USA, 004092720), 25 μg / mL hydrocortisone sodium succinate (Pfizer Inc, 00009001305), 2 mM L-glutamine (Thermo Fisher Scientific, Waltham, MA, USA, 25030081) and 5% Antibiotic-Antimycotic solution (Thermo Fisher Scientific, 15240096) were added; L-glutamine, insulin, hydrocortisone and L- glutathione were all added to the solution no more than 24 h prior to use. Liver procurement: 10–12-week-old male Lewis rats in the weight range of 250–300 g (Charles River Laboratories, Wilmington, MA, USA) were used for all experiments to ensure comparable results between groups. All animals were socially housed in controlled, standard conditions (21 °C, 12-h light / dark cycle, 30–70% humidity, mixed paper / cellulose bedding, pathogen free HEPA filtered ventilated cages). The rats had unrestricted access to sterile water and chow, in accordance with National Research Council Guidelines. Rats were cared for by the Massachusetts General Hospital (MGH) Center for Comparative Medicine (CCM). Considering the prolonged duration of the experiment rendered the organ more prone to contamination, all following surgical procedures were carried out using sterile technique, consumables and solutions. Surgical procurement was performed as described in Berendsen et al., Transplant Res. 2012;1:6. Donor rats were anesthetized under 5% isoflurane. The abdomen was opened via a transverse abdominal incision. Ligaments connecting the superior and inferior portions of the liver were dissected and the portal vein was exposed. The gastric and splenic branches of the portal vein, Attorney Docket No. 29539-0832WO1 / MGH 2024-313 as well as the hepatic artery were ligated using 6-0 silk. The bile duct was partially dissected, cannulated using 24 g tubing, and secured with 6-0 silk. The inferior vena cava was heparinized with 1 U / g using a 30-gauge insulin syringe. Following 5–10 min of heparin circulation, the portal vein was cannulated with a 16 g cannula, followed by transection of the inferior vena cava (IVC). The cannula was connected to 16 g tubing attached to a 60 mL syringe containing 1 mL heparin in 60 mL saline. The portal vein was hand flushed at 10 mL / min for 4 min, after which the remaining connective tissue was dissected, and the liver was removed from the body cavity. Following removal, the liver was flushed with the remaining 20 mL saline, immediately weighed and connected to the perfusion system, keeping warm ischemic time below 5 min. Normothermic machine perfusion and viral treatment: Perfusate circulation was carried out as described in Mojoudi et al., Heliyon. 2024;10:e29519. Using a roller pump system (Masterflex L / S, Vernon Hills, IL, USA) with two separate sets of tubing delivering perfusate into and out of the perfusion reservoir. The system was consistently kept at a temperature of 37 °C via a water bath (PolyScience, Niles, IL, USA) continuously pumping heated water through the double-jacketed perfusion system components (Radnoti, Covina, CA, USA). Perfusate oxygen concentration was maintained within a close range of 500 mmHg using a 95% O2 / 5% CO2gas cylinder (Airgas, Radnor, PA, USA). System pressure was zeroed, the liver was placed in the tissue bath and connected to the system. Flow rate was brought from 5 mL / min to 30 mL / min gradually, maintaining a maximum portal pressure of 11 mmHg and minimum flow rate of 20 mL / min throughout the perfusion. The viral particles (3 × 107IU) were injected manually into the bubble trap at 30 min, at which time the system contained 200 mL total volume of perfusate. Every hour 50 mL of perfusate was added. At 12 h, a complete switch of perfusate was performed to a total volume of 200 mL and a second viral injection (3 × 107IU) took place. Overnight, 100 mL of perfusate was added every 2 h until 24 h was reached, and the perfusate was completely switched to perfusate with a total volume of 500 mL. Subsequently, 3-hourly replacements of perfusate were carried out until the end of perfusion (72 h). All perfusate switches were performed in a gradual fashion, perfusing the liver with incremental combinations of new and old perfusate to prevent potential damage, until Attorney Docket No. 29539-0832WO1 / MGH 2024-313 the perfusate was completely switched to circulate the new perfusate. In accordance with the timeline outlined in the study design (FIG. 2A), outflow samples were collected directly from the basin in close proximity of the inferior vena cava, while inflow samples were collected from a port placed above the cannula perfusing the portal vein. The liver was weighed following upon the end of perfusion to determine weight change. Biopsies of the left lateral lobe (LLL) and right medial lobe (RML) were carried out immediately after, with the LLL sample being snap frozen in liquid nitrogen for subsequent ATP analysis and the RML sample being formalin-fixed for histological analysis. The liver was then stored in cold saline and transported for imaging. Liver viability assessment: According to the time points established in the study design (FIG. 2A), inflow and outflow perfusate samples were analyzed using a Siemens Rapidpoint 500 (Siemens, Munich, Germany). Liver performance metrics were analyzed to determine liver functionality during perfusion (pH, O2consumption, lactate clearance, potassium). Following each experiment, collected outflow samples were analyzed for hepatic injury markers ALT and AST enzymes using a Piccolo Xpress (Abaxis, Union City, CA, USA). Portal resistance was calculated using pressure readings taken at every time point, and defined as: pressure / flow. initial weight. Oxygen consumption was defined as: (inflow partial pressure of O2- outflow partial pressure of O2) oxygen solubility coefficient / initial weight. Weight change was defined as: final weight - initial weight / initial weight, presented as a percentage. VCA Procurement and Transplantation Rat hindlimbs were used as model vascularized composite allografts (VCAs). After induction using isoflurane (5%) inhalation with 100% O2, general anesthesia was sustained with inhaled isoflurane (1–3%) and anesthesia depth was confirmed with a toe pinch test. Hindlimbs were procured as described in Sucher et al., Vis Exp. 2010 Jul 12;(41):2022. Briefly, grafts included the knee joint with 10 mm distal femur, along with thigh muscle groups with the inguinal fat pad and the lower leg and foot. Femoral vessels were skeletonized and ligated 5 minutes after IV administration of 100 IU / mL / kg heparin in the penile dorsal vein. The femoral artery was cannulated with a 24 G angio catheter and secured with a 6 / 0 nylon suture. The femoral vein was cut after ligation. Immediately after procurement, a pressure-controlled manual flush Attorney Docket No. 29539-0832WO1 / MGH 2024-313 (< 60 mmHg) with 3 mL (200 IU) of heparin saline at room temperature was performed. After perfusion, the VCA was transplanted into a recipient. For the non- rejection (syngeneic) model, Lewis animals (rats) were both donor and recipient; for the partial mismatch rejection (allogeneic) model, Lewis animals acted as donor, Fischer 334 animals as recipient; for the full mismatch rejection model, Brown Norway animals acted as donor, Lewis animals as recipient71. Recipient vessels were prepared on the contralateral side in a similar fashion to the donor. Vessels were ligated distally and prepared for anastomosis. For the partial hindlimb (heterotopic) transplant model72, the foot was removed prior to implantation. A circular incision was made proximal to the ankle joint, the vessels and muscles are ligated using cauterization. The bone was transected proximal to the tibio-fibular syndesmosis. A longitudinal incision in the flank was made with subsequent tunneling to the groin area for VCA insertion. Femoral arteries and veins were anastomosed using a self- developed adjusted cuffing technique to allow for application to partial hindlimb transplant. Skin on the donor VCA was excised to create an oval flap in the flank which was secured with interrupted 5-0 sutures. Inguinal fat pad and groin skin incision were similarly closed with interrupted 5-0 vicryl sutures. For the whole limb (orthotopic) transplant model70, circular skin incision was made on the thigh. The existing hindlimb was removed in similar fashion to the donor animal, however after ligation of the arterial and venous branches, ligation of the femoral vessels takes place distally, as described for the partial hindlimb model. Femur was connected by inserting a pin (18 G needle tip) in the donor and recipient. Femoral arteries and veins were anastomosed using the traditional cuffing technique. For long-term observation, some animals underwent additional sciatic and femoral nerve reconnection using 10-0 standing sutures. Muscle and skin were approximated using 5-0 vicryl interrupted and continuous sutures, respectively. Rejection animals that underwent immunosuppression (IS) were treated with Cyclosporine A (10 mg / kg, SQ) from post- operative day (POD) 2 onwards. Normothermic Machine Perfusion (VCA) Perfusion Parameters: Modified Steen (Steen+) was used as perfusate and prepared as described in Goutard et al., J Reconstr Microsurg. 39, 350-360 (2023), with minor adjustments of the sodium chloride and sodium bicarbonate, which were Attorney Docket No. 29539-0832WO1 / MGH 2024-313 decreased from 86 mmol / L to 84 mmol / L and from 15 mmol / L to 14 mmol / L, respectively. Furthermore, 100 mg / L of vancomycin and ceftriaxone was added. Perfusate was circulated using a roller pump system (Masterflex L / S, Vernon Hills, IL) with two separate sets of tubing (Masterflex platinum-cured silicone tubing, L / S 13, Cole-Parmer, Vernon Hills, IL) delivering perfusate into and out of the perfusion reservoir. Temperature was regulated by a water bath (Polystat Cooling / Heating Circulating Bath, Cole-Parmer), set at 37 °C, through double-jacketed perfusion system components (Radnoti, Covina, CA, USA). Perfusate oxygen concentration was maintained within a close range of 450 mmHg using a 95% O2 / 5% CO2gas cylinder (Airgas, Radnor, PA, USA). Pressure transducer (PT-F, Living Systems Instrumentation, St Albans City, VT) was connected close to the angio catheter (BD Angiocath 24G) in the femoral artery during perfusion. Prior to start of the perfusion, base pressures were obtained to account for the system pressure of each flow rate. A maximum flow of 1.5 mL / min was observed, and flow was regulated in a pressure- based manner, aiming at a vascular resistance of 30-35 mmHg. Viral Treatment: Transduction enhancer protamine sulfate (20 μg / mL of perfusate) for the 12 h perfusions and polybrene (10 μg / mL of perfusate) for the 6 h perfusions were added to the viral particles, prior to injection. Viral injection (1.5 x 109IU) took place 1 h after the start of perfusion (T0) for the 12 h perfusions and at 45 min for the 6 h perfusions, as to enable stabilization of the resistance within the graft prior to injection. Injection was done through the bubble trap in the 12 h perfusions, and through an inflow port close to the arterial cannula in the 6 h perfusions. A minimum flow of 0.3 mL / min of Steen+ was maintained during injection to avoid backflow, and a maximum vascular pressure of 40 mmHg was observed. Following injection, a closed circuit was created with a total volume of 220 mL of perfusate. Approximately every 2 h, all the perfusate circulated the system. At 11 h or 5 h 45 m respectively, the circuit was opened and fresh perfusate was used to wash out the intraluminal viral particles. Hence, during the 12 h and 6 h perfusions there was 10 h and 5 h of viral circulation time, respectively. For the perfusions with the constitutive promoter, appropriate control groups were determined as shown in FIG. 14C. Attorney Docket No. 29539-0832WO1 / MGH 2024-313 Viability Assessment: Inflow and outflow perfusate samples were analyzed using a Siemens Rapidpoint 500 (Siemens, Munich, Germany). VCA performance metrics were analyzed to determine viability during perfusion (pH, O2consumption, glucose consumption, lactate, potassium). NaHCO3- titration was performed to correct for acidosis if needed. Vascular resistance was calculated using pressure readings taken at every timepoint, and defined as: pressure / flow*initial weight. Oxygen consumption was calculated using a modified Fick equation using circuit flow, limb weight, and pre- and post-limb oxygen contents (0,00314*Flow(pO2in – pO2out) / weight). Weight change was defined as: final weight - initial weight / initial weight, presented as a percentage. Intramuscular Viral Delivery (VCA) Hindlimbs of animals (Lewis, male, 250 ± 25 grams, Charles River Laboratories, Wilmington, MA) were injected with 6 x107IU or with sterile saline (2 mL), half of which was injected intramuscular (i.m.) and half subcutaneous (s.q.) (n=1 per group). In a partial hindlimb transplant model, two further hindlimbs were procured and injected with 5 x 108IU (15 mL), half of which was injected i.m. and half intravenous (i.v.). One of the limbs was flushed with saline and transplanted after 1 h warm ischemic time (WIT), and one of the limbs was subjected to 6 h static cold storage (SCS) to extend the dwell time of the virus, before flushing and transplanting the limb. Both transplants were followed up by clinical observation as well as blood draws on POD 0, 1, 3, 7, 14 or until graft failure occurred. Quantification of GLuc in Tissue and Viral RNA in Perfusate Post-perfusion, tissues were pulverized, homogenized and protein and nucleic acids were extracted from each lobe using commercial kits (Nanolight Technology, Pinetop, AZ, USA, GLuc FLASH Assay, and Qiagen, Hilden, Germany RNeasy Kit, respectively). GLuc substrate assay: GLuc substrate, native coelenterazine enzyme (Nanolight Technology, 303), was reconstituted at 2.7 mg / mL in 200-proof ethanol. Working solutions were made fresh, immediately prior to assay with a 1:1000 dilution in PBS. Twenty microliters of samples (plasma, cell supernatant, perfusate) were added to black-walled, clear-bottom 96-well plates and 100 uL of CTZ solution and immediately read using a bioluminescent plate reader (Varioskan, Thermo Fisher). Attorney Docket No. 29539-0832WO1 / MGH 2024-313 All samples were read forward and in reverse to account for any time-dependent signal degradation. The concentration of GLuc in each sample was calculated by taking the average of the forward and reverse readings. Assays were performed under controlled lighting conditions with minimal exposure, and the temperature was maintained consistent throughout the experiments. GLuc Quantification: RNA Isolation & qRT-PCR: Total viral RNA in perfusate samples and tissue lysates was assessed via qPCR using a lentivirus titration kit (Applied Biological Materials, Richmond, Canada, LV900). In brief, tissue samples were pulverized, homogenized and RNA was extracted from each tissue sample using a commercial kit (RNeasy Kit, Qiagen). RNA concentration and quality were checked using NanoDrop One (Thermo Scientific). Equal amounts of total RNA from each sample were pooled and used for quantitative real-time PCR (qRT-PCR). GLuc expression was determined using the primers FWR: 5'- CACGCCCAAGATGAAGAAGT-3’ (SEQ ID NO:5), REV: 5- 'GAACCCAGGAATCTCAGGAATG-3’ (SEQ ID NO:6) and probe 5-' / 56- FAM / TACGAAGGC / ZEN / GACAAAGAGTCCGC / 3IABKFQ / -3' (SEQ ID NO:7) in a Viia 7 Real-Time PCR System equipment (Thermo Fisher Scientific). Beta-actin (Actb) was also measured as an endogenous reference gene (TaqMan Gene Expression Assays, Rn00667869_m1) and used in the comparative cycle threshold method (DDCt) to determine relative gene expression. GLuc Quantification: Protein Extraction: Protein was extracted from pulverized and homogenized tissue samples using a commercial kit (GLuc FLASH Assay, Nanolight Technology). Lysates were centrifuged (10min, 3,000g) and pelleted debris were discarded. GLuc was then quantified in the supernatant of each sample via GLuc Assay, as described herein. Total protein was determined using a Pierce™ BCA Protein Assay Kit (Thermo Fisher) and used to normalize GLuc values in each sample. Bioluminescent and Fluorescent Imaging Liver: Both the perfused liver and a freshly procured control liver were injected with 4 μg / g of liver of colentazerine prior to luminescence imaging. Bioluminescence imaging was carried out using the Spectral AMI X (Spectral Instruments Imaging Optical Imaging Platform) and Sapphire NIR fluorescent Attorney Docket No. 29539-0832WO1 / MGH 2024-313 scanner (Azure Biosystems, Dublin, CA, USA) at the Center for Systems Biology Core (MGH, Boston, MA, USA) to visualize the expression of GLuc. For signal intensity analysis, background radiation was subtracted. After imaging, the liver was dissected by liver lobe and stored at −80 °C for later quantitative analyses of GLuc and viral DNA. VCA (hindlimbs): Animals that received transduced and non-transduced grafts were injected with 4 μg / g bodyweight of Colentazerine (Nanolight, Colentazerine- INJ, Cat. N. #303) prior to luminescence imaging. Bioluminescence imaging was carried out using the Spectral AMI X (Spectral Instruments Imaging Optical Imaging Platform) at the Center for Systems Biology Core (MGH, Boston, MA, USA) to visualize expression of Gaussia Luciferase. For signal intensity analysis, background radiation was subtracted. After imaging, the VCA was dissected into the sections illustrated in FIG. 15D with every section being stored at -80°C for later quantitative analyses of Gaussia Luciferase and viral DNA. Histological Analysis Tissue sections were stained with hematoxylin-eosin (H&E), Terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL), and Periodic acid- Schiff (PAS) (Specialized Histopathology Services Core, MGH, Charlestown, MA, USA). Microscopic analysis was performed by blinded pathology assessment. Assessment of VCA Graf-Specific Transduction Characteristics Primary Cell Isolation: Four hindlimbs (n=2, per group) were procured in sterile fashion (Lewis, male, 250±25 grams, Charles River Laboratories, Wilmington, MA) and submerged in ice-cold HBSS with 5% P / S. Skin, muscle and endothelial tissues were dissected in a sterile hood in order to isolate keratinocytes, fibroblasts, myocytes and endothelial cells. Briefly73, for keratinocytes and fibroblasts, skin tissuewas excised and any subcutaneous fat was removed and incubated overnight at 4 C inCollagenase Type IV (Thermo Fisher) reconstituted at 10 mg / mL in PBS. The following morning, the epidermis and dermis was separated. Keratinocytes were released from the epidermis through gentle agitation and filtered through a 40 m cell strainer (Sigma Aldrich). The keratinocytes were then spun down at 1,100 x g for 10 minutes and then resuspended in freezing media consisting of 90% Keratinocyte Attorney Docket No. 29539-0832WO1 / MGH 2024-313 Medium (Sigma Aldrich, supplemented with 1% P / S) and 10% DMSO and cryopreserved in liquid nitrogen until further analysis. To isolate fibroblasts, the dermis was minced and then further digested for 1.5 h in Collagenase Type Ireconstituted at 1 mg / mL at 37 C. Afterwards, the fibroblasts were released from thedermis through gentle agitation and filtered through a 40 m cell strainer. The fibroblasts were then spun down at 1,100 x g for 10 minutes and then resuspended in freezing media consisting of 90% DMEM / F12 (Thermo Fisher, supplemented with 10% FBS, 1% Glutamine, and 1% P / S) and 10% DMSO and cryopreserved in liquid nitrogen until further analysis. To isolate myocytes, skeletal tissue was excised and any connective tissue was removed. The tissue was then minced and digested using an enzymatic solution of dispase 2 (Sigma Aldrich, 0.6 U / mL) and Collagenase D (Sigma Aldrich, 2.5 mg / mL) reconstituted in PBS at 37 C. Following digestion, the tissue was passed through a 100 m filter (Sigma Aldrich), spun down at 1,100 x g for 10 minutes and then resuspended in red blood cell lysis buffer (Promega) and DMEM (supplemented with 10% FBS and 1% P / S) at a 7:1 ratio and filtered through a 40 μm cell strainer. The myocytes were then spun down at 1,100 x g for 10 minutes and then resuspended in freezing media consisting of 90% DMEM (supplemented with 10% FBS and 1% P / S) and 10% DMSO and cryopreserved in liquid nitrogen until further analysis. For endothelial cell isolation, the femoral artery and vein were excised, and the vessels were each dissected in a single longitudinal plane. The vessel was then placed in a petri dish with endothelial layer facing down and cultured in endothelial growth medium 2 (Lonza, EGM) at 37 °C and 5% CO2until endothelial sprouting was observed. Following sprouting, the vessel was removed and the isolated endothelial cells were cultured to confluency. At confluency, the endothelial cells were detached using trypsin (Thermo Fisher), spun down at 1,100 x g for 5 minutes and then resuspended in freezing media consisting of 90% EGM and 10% DMSO and cryopreserved in liquid nitrogen until further analysis. Lentiviral Transduction: Primary rat cells (vascular endothelial cells, fibroblasts, myocytes and keratinocytes) were seeded at 30% confluence in 24-well plates and allowed to adhere overnight in respective complete media (Thermo Fisher) with 10% FBS (GIBCO) and 1% antibiotic / antifungal. EF1a-GLuc-IRES-RFP lentiviral vectors were added at different MOIs to each well in a total volume of 1 mL Attorney Docket No. 29539-0832WO1 / MGH 2024-313 using antibiotic-free medium. 10 μg / mL of the transduction enhancer Polybrene (Sigma-Aldrich) was also added to each well. Cells were incubated for 24 h before medium was changed to complete medium (unless otherwise noted). Transductions were carried out at 37 °C (unless otherwise noted). Efficiency was determined 72 h post-transduction: plates were scanned for RFP fluorescence imaging and quantification using Celigo® Imaging Cytometer (Nexcelom Bioscience, Lawrence, MA); 300 μL of supernatant were collected from each well for GLuc assessment. Cell viability was determined via automated cell counter (NucleoCounter®). Viral Uptake Rate: Human umbilical vein endothelial cells (HUVECs) were plated in 24-well plates at 5x104cells / well in EGM™-2 Endothelial Cell Growth Medium-2 BulletKit™ (Lonza Bioscience). LV vectors were added at MOI 200 with 10 μg / mL of Polybrene and each group was exposed to viral vectors for increasing amounts of time (30 min, 2 h, 4 h, 8 h and 24 h). Control groups were empty wells with no seeded cells that received the same concentration of viral vectors. At the end of viral exposure time, fresh medium (EBMTM-2 Endothelial Cell Growth Basal Medium-2 - Lonza Bioscience) was added to each well. 72 h later, 300 μL of supernatant was aliquoted for p24 ELISA (Takara Bio USA, Inc) assessment and determination of viral uptake rate. Plates were also imaged for RFP fluorescence. In Vitro Validation of Inducible Promoter (VCA) Blood Plasma Collection: To obtain plasma from rejecting animals, two animals were transplanted in a full mismatch (Brown Norway to Lewis) and non- rejection (Lewis to Lewis) model as control (male, 250 ± 25 grams, Charles River Laboratories, Wilmington, MA). Partial hindlimb transplants were performed, and rejection was clinically and histologically observed. At POD 7, cardiac puncture was performed in sterile fashion and plasma was obtained for in vitro stimulation studies. In Vitro Validation Studies: HEK293T (ATCC) cells were transduced with lentiviral vectors expressing either NF-kB-GLuc-IRES-mRFP or EF1a-GLuc-IRES- RFP (control group). The transduction protocol followed steps similar to those outlined in Lentiviral Transduction as detailed herein. Postoperative Assessments (VCA) Postoperatively, daily flap images were taken for clinical assessment by an experienced clinician. Blood draws were obtained as described in Charlès et al., J Vis Attorney Docket No. 29539-0832WO1 / MGH 2024-313 Exp. 2023 Jun 30;(196):10.3791 / 65513 on PODs 1, 3, 5, 7 and 9, and skin and muscle were obtained of two replicates per group, with 3 mm biopsy for histology and 5 mm biopsy punch for RT-qPCR. Whole blood was centrifuged, and plasma was stored at - 80°C until assessment. At sacrifice, vessel patency was confirmed using the milking test (see, e.g., Podeur et al., Head Neck. 2019 Sep;41(9):3328-3333). Blood was obtained by cardiac puncture, and tissue was obtained from the transplant (skin, muscle, vasculature), and from off-target organs (heart, lung, liver, spleen, kidney, intestine, brain) for histological and RT-qPCR analysis in all replicates. Biopsies were fixed in formalin and processed for histopathological examination and flash frozen for RT-qPCR analysis. Slides were stained with hematoxylin and eosin (H&E). Banff Score: Blinded evaluation by a pathologist was performed for all biopsy samples using the Banff score criteria to assess acute cell-mediated rejection in skin tissue75,76. Briefly, grade 0 is considered no rejection, grade I mild (mild perivascular infiltration, no involvement of epidermis), grade II moderate (moderate perivascular infiltration, possible mild epidermal involvement), grade III severe (dense inflammation and epidermal involvement) and grade IV necrotizing acute rejection (frank necrosis of the epidermis and other skin structures). Muscle tissues were evaluated and scored using the histology injury scoring system (HISS) for hypoxia- induced muscle injury77. Plasma Cytokine Assessment: Cytokine arrays on rat plasma samples were performed by Eve Technologies (Calgary, Alberta, Canada), according to their protocol. Rat Cytokine / Chemokine 27-Plex Discovery Assay® Array - RD27 measured the following cytokines, chemokines and growth factors in the same sample: Eotaxin, EGF, Fractalkine, IFNγ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17A, IL-18, IP-10, GRO / KC, TNFα, G-CSF, GM-CSF, MCP-1, Leptin, LIX, MIP-1α, MIP-2, RANTES and VEGF-A. Statistical Analyses Statistical analyses were performed with Prism 10 software Version 10.2.3 (Graphpad Software, San Diego, CA, USA, graphpad.com) with a two-sided significance level of 0.05. One-way analysis of variance (ANOVA) was performed to analyze independent variables with three or more categorical groups, while Two-way ANOVA was performed to compare multiple groups, followed by Tukey’s post-hoc Attorney Docket No. 29539-0832WO1 / MGH 2024-313 test to examine statistical differences. For pairwise comparisons, unpaired T-test was performed to compare groups. Metrics were reported as mean with range as error bars. For specificity and sensitivity analyses of biomarkers, receiver operating characteristic (ROC) curves were generated, and the areas under the curve (AUC) were used to assess statistical differences. The "time-to-diagnose probability" curve was analyzed using a Kaplan-Meier estimate. Example 1: Lentiviral vector design and validation Lentiviral vectors were designed with the constitutive promoter EF1α driving the expression of the secreted biomarker GLuc and the fluorescent tag RFP (FIG. 1A). In vitro validation of the genetic construct was carried out with human hepatocytes (HepG2 cells), which were treated with increasing doses of viral vectors (MOI). Engineered cells exhibited a clear dose-dependent expression of the fluorescent marker, which was used to calculate transduction efficiency (FIGS. 1B, 1C). Cell viability, however, exhibited an inversely proportional relationship to MOI, suggesting that higher viral doses can be toxic to hepatocytes. These findings guided our decision regarding viral dosing for the subsequent ex vivo liver perfusions. Considering there are approximately 300 million hepatic cells in a rat liver103, using an MOI of 100 would require about 3 × 1010IU. Based on our earlier findings, we believed this dose would be excessively high and potentially toxic to the liver. Since hepatocytes are generally easy to transduce, we performed initial perfusions with a 1000 times lower dose (3 × 107IU). Example 2: Perfusion parameters showed comparable viability between groups Rat livers were procured with a warm ischemic time of less than 5 minutes, ensuring minimal damage prior to perfusion. The overall average initial weight was 11.3 ± 1.3 g, indicating uniformity in the sample set. Next, livers were connected to the machine perfusion system and perfusion was started (FIG. 1D). Livers were exposed to viral particles for 24 h, after which they were perfused for another 48 h without viral particles until 72 h of perfusion was reached (FIG. 1E). Due to a technical failure, one liver reached 48 h instead of 72 h, results of which are shown in the biomarker analysis. Attorney Docket No. 29539-0832WO1 / MGH 2024-313 Perfusion parameters were monitored to ensure stability and comparability between the experimental and control group. Prior to the viral injection time point (30 min), a stable flow of 30 mL / min was reached in all replicates of both the experimental and control group. Vascular resistance remained stable for 65 h in all replicates and remained between 0–3 mmHg / mL / min throughout all perfusions. Further perfusion parameters are displayed in FIGS. 2A-2E. Potassium remained between physiological levels of 4–6 mmol / L for both groups (FIG. 2B). Oxygen consumption remained stable throughout perfusion in both groups (FIG. 2D), suggesting effective maintenance of metabolic activity. Lactate accumulation was apparent during the first 24 h to maximum levels of 3.99 mmol / L (FIG. 2C), which was the extended closed-loop phase for viral recirculation. However, clearance was apparent, especially between 12 and 24 h in the viral group. After 24 h, viral exposure was ended and perfusate switches were performed every 3 h. During this time, lactate clearance was apparent in both groups and lasted until the end of study, indicating functional metabolic processes. Bile production lasted until at least 42 h with some replicates producing bile until the end of study (FIG. 2E). Energy charge assessment indicated low but comparable levels in both groups. Example 3: Bioluminescent and fluorescent imaging demonstrated successful transduction At the end of perfusion period, livers were subjected to bioluminescent and fluorescent imaging to assess transduction success. Ex vivo bioluminescent imaging showed a remarkable mean 8000-fold increase in luminescence of the transduced livers compared to controls. Signal intensity seemed to be highest at the portal vein which is where the perfusate enters the liver (FIG. 3A). Furthermore, central regions, which generally were well-perfused and have higher tissue thickness, showed higher signal intensity than peripheral regions. Quantitative analysis confirmed significantly higher luminescence in the experimental group (FIG. 3B, p < 0.0001). For reference, the liver that was perfused for 48 h due to a technical failure is also shown and demonstrated a signal comparable to the control group. Fluorescence quantification, while lower, still showed higher fluorescence in the viral group (FIGS. 3C, 3D). These results collectively verified the effectiveness of the lentiviral vectors in achieving successful gene expression in the liver tissue. Attorney Docket No. 29539-0832WO1 / MGH 2024-313 Example 4: Biomarker analyses verified transgene expression in all liver lobes To further confirm transgene expression, perfusate and tissue samples were analyzed for viral RNA and GLuc presence (FIGS. 4A-4D). Perfusate analysis revealed a clear increase in viral RNA after injection of the viral particles, followed by steady decline over the first 24 h suggesting uptake of the viral particles by the liver. After the perfusate switch at 24 h, the viral RNA returned to pre-injection levels, suggesting successful clearance of the viral particles which persisted until the end of perfusion. A similar trend was seen in the bile secretion. Tissue analysis showed a significant increase in GLuc signal at 72 h (p < 0.0001). Conversely, at 48 h no significant GLuc levels were found (FIG. 4B), highlighting the importance of the full 72 h period for effective transgene expression. Assessment of GLuc per liver lobe showed homogenous distribution throughout the lobes with a similar column factor (p < 0.0001). Concurrently, tissue analysis of GLuc mRNA via RT-qPCR revealed transgene expression in the experimental group, while no GLuc transgene expression was found in the 48 h liver (technical failure replicate) nor the control group (FIG. 7). Example 5: Histological findings were comparable between groups Histological analysis at the end of perfusion revealed similar levels of apoptosis between transduced and control livers. Interstitial edema and vacuolization were observed to be variable within groups but remained comparatively similar between groups (FIGS. 5A-5F). These findings suggested that the lentiviral transduction process did not induce additional histological damage beyond what was observed in the control group, indicating a level of safety and feasibility for this approach. Example 6: Genetically engineered organs exhibited sustained transgene expression beyond 300 days This study featured an innovative platform for genetically engineering reporter VCAs utilizing rat hindlimbs as a working model. FIGS. 9A-9F illustrate the overall protocol, starting with procured grafts undergoing ex vivo machine perfusion. Preservation of VCAs with perfusion was leveraged as a local delivery platform for lentiviral vectors expressing genetic constructs of interest. Perfused VCAs were transplanted into recipient animals, enabling the in vivo detection of transgene Attorney Docket No. 29539-0832WO1 / MGH 2024-313 expression by postoperative day (POD) 3. Further monitoring of transplanted animals was conducted postoperatively through blood and tissue analyses. To establish proof-of-concept, we first developed a construct featuring a robust, constitutive promoter (elongation factor 1-alpha, EF1α), driving the expression of a sensitive secreted biomarker, Gaussia Luciferase (GLuc), alongside a red fluorescent tag, RFP (FIG. 13A). Different viral delivery routes were initially evaluated to ensure optimal and effective tissue transduction. Systemic intravenous (i.v.), and local i.v., intramuscular (i.m.), and subcutaneous (s.q.) viral delivery routes were ineffective and even led to graft failure in the transplant model (FIGS. 13B, 13C). Hence, a machine perfusion protocol was developed to ensure homogenous viral delivery while maintaining graft viability (FIGS. 14A-14E). VCA-specific transduction parameters were characterized in vitro using relevant cell types for the model, to further refine the perfusion parameters. Rat-derived primary fibroblasts were transduced with increasing viral multiplicities of infection (MOI), revealing a directly proportional relationship between transduction efficiency – measured as a function of GLuc secretion – and dose (FIG. 14D). As the viral load increased, however, cell viability was somewhat affected, suggesting that there was a threshold beyond which the viral dose becomes toxic and affects VCA cell viability. Viral transduction was then performed at different temperatures to assess the optimal perfusion temperature (normothermic22at 37 °C, subnormothermic7at 21 °C and hypothermic28,29at 4 °C). The physiological temperature of 37 °C yielded the highest transduction efficiency, supporting the use of normothermic machine perfusion protocol (FIG. 14E). Viral levels were monitored throughout perfusion, confirming successful flush-out at the end as evidenced by viral RNA returning to pre-injection baseline levels (FIG. 9B). Other critical parameters were monitored during perfusion (i.e., potassium, lactate, glucose and oxygen consumption), suggesting stable viability in both control and viral groups (FIG. 15A). The mean weight gain was within transplantable levels in both groups, as outlined by Goutard et al.30and Charles et al.31. Postoperatively, VCAs from both groups exhibited similar clinical evolution (FIG. 15B). In vivo bioluminescence imaging confirmed successful transduction of virally perfused animals (FIG. 9C), revealing an approximately 10-fold increase in Attorney Docket No. 29539-0832WO1 / MGH 2024-313 total luminescence at the transplant site (FIG. 9D). Similarly, GLuc plasma levels confirmed successful transgene expression, with a peak of secretion between PODs 3 and 7, followed by a decline after POD 14. The signal then stabilized for the remainder of the postoperative period, to date, POD 209 (FIG. 9E). Finally, tissue analysis revealed GLuc mRNA expression in the flap tissues, showing over a 15-fold increase in the vasculature compared to control tissues (FIG. 9F). This indicated the highest transduction levels in the vascular endothelium, suggesting its role as the main 'gateway' for viral vectors during organ perfusion, followed by skin and muscle. FIG. 15C provides a more detailed tissue analysis, confirming no evidence of transduction in off-target tissues. Sectional analysis of the skin flap revealed a relatively uniform distribution of transgene secretion, indicating homogeneous transduction throughout the tissue (FIG. 15D). Histopathological analysis at the end of the study revealed necrosis in skin tissue of some replicates in both groups, characterized by epidermal loss, apoptotic bodies, and cell infiltration. Additionally, ischemic changes in muscle tissue were observed, including myocyte damage and interstitial edema, indicating overall compromised viability of the grafts (FIG. 15E). Therefore, we optimized the machine perfusion protocol to enable transduction within the 6 h preservation time limit for VCAs, thereby avoiding the tissue damage observed with 12 h perfusions. In parallel, the transplant model was also optimized to the more clinically relevant orthotopic VCA model (see Materials and Methods), which was technically less challenging and had shorter surgical warm ischemic time. Based on previous in vitro findings (FIG. 14D), the viral dose was increased to 1.5 x 109infection units (IU) for 6 h perfusions. Parameters showed low resistance throughout the perfusion period, physiological levels of potassium and lactate, even during the closed-circuit phase, and stable oxygen and glucose consumption. At the end of perfusion, weight gain was within expected range, showing no significant differences between viral and control groups, suggesting good post-perfusion viability (FIG. 16A). Clinical images demonstrated excellent recovery with normal hair growth (FIG. 16B). Histopathological analysis confirmed good tissue viability with a maximum muscle injury score of 6 limited to the immediate postoperative period, and skin Banff scores of 0 (see Materials and Methods) throughout the entire postoperative period (FIG. 16C). In vivo bioluminescent Attorney Docket No. 29539-0832WO1 / MGH 2024-313 imaging showed an approximately 4-fold increase compared to control on both POD 7 and POD 28 (FIG. 16D). Example 7: Graft-specific transduction: characterization of endothelial viral uptake and transduction rates Building on our observations of the role of the vasculature in VCA viral transduction, we aimed to further characterize this phenomenon in vitro. We compared several primary rat cells to evaluate cell-specific transduction efficiency. Vascular endothelial cells exhibited significantly higher levels of transgene expression, corroborating the observations made in vivo (FIG. 10A). Fibroblasts, myocytes, and keratinocytes exhibited similar transduction levels, which were directly proportional to the duration of viral exposure. Importantly, transduction was only detectable after 48-72 h, which again aligns with in vivo observations and known lentiviral transduction characteristics reported in the literature16,32-34. Next, we sought to characterize and model the viral uptake rate by endothelial cells over time. Viral uptake significantly peaked between 8 h and 24 h of exposure, reaching a rate of approximately 7.25 ng / mL / 106cells (FIG. 10B). Interestingly, after 72 h of incubation, transduction was observed even in cells with as little as 30 min of viral exposure (FIG. 10C). As expected, transgene expression – measured as a function of RFP intensity – increased with longer viral exposure times, peaking between 8 h and 24 h (FIG. 10D). Notably, endothelial cells appeared inactivated by morphology in all tested groups, indicating that lentiviruses did not elicit an injury response from these cells. These observations confirmed the temporal dynamics of viral uptake and transgene expression as well as suggesting that endothelial cells could sustain lentiviral exposure without triggering an activation response. These findings can be particularly relevant for assessing potential interventions and refining gene delivery systems designed for whole-organ applications. Example 8: Biosensor cells reported rejection in vitro with high sensitivity and specificity Genetic biosensors are an exciting technology that have been applied to many areas of diagnostics, but not often evaluated in the context of whole organ transplantation. By designing an inducible genetic construct, we aimed to generate Attorney Docket No. 29539-0832WO1 / MGH 2024-313 engineered cells that can be switched on to secrete a biomarker in response to specific cues. To that end, we designed an inflammation-responsive promoter region, featuring two repeats of the nuclear factor kappa B (NF-kB) response element followed by the minimal Tyrosine Kinase (TK) promoter (FIG. 11A). NF-kB is a key transcription factor that plays a central role in regulating multiple inflammatory events associated with organ rejection35-37. We leveraged its function as a master transcriptional activator to drive the local expression of GLuc and RFP under rejection settings. First, a proof-of-concept engineered cell line expressing the synthetic genetic construct was generated and stimulated with the pro-inflammatory cytokine TNF- α, a gold-standard stimulator of the NF-kB signaling pathway (FIG. 11B). Cells were exposed to stimulation for progressively longer periods and exhibited a directly proportional response to activation, as indicated by biomarker secretion in the supernatant (FIG. 11C). The cessation of stimulation led to a corresponding interruption in GLuc secretion, demonstrating that the construct could be toggled on and off in response to environmental stimuli (FIG. 11B). Cells were subsequently exposed to POD 7 serum from both rejection and non-rejection animals (FIGS. 17A-17C). FIG.11D validated the specificity of the NF-kB sensor when exposed to plasma from rejecting transplanted rats (detected as RFP expression) when compared to non-rejecting plasma exposure. Cells expressing the same genes under the control of the ubiquitous promoter EF1a were used as controls, exhibiting no transcriptional inducibility. Further supporting these observations, cells treated with rejection plasma exhibited significantly higher supernatant GLuc levels after 24 h of exposure (FIG. 11E). Notably, POD 7 plasma of non-rejection animals exhibited lower inflammatory cytokine levels compared to rejection group, but as expected, still significantly higher than those of healthy, non- transplanted animals given the early postoperative phase (FIG. 17D). This suggested that the inflammatory milieu of non-rejection animals was not sufficient to activate the inducible synthetic system, indicating that the signal strength needed to reach a threshold for proper activation. This implied that our platform exhibited the desired dose-response characteristics to specifically respond to local rejection events. Finally, ROC curves were used to evaluate the performance of GLuc and TNF-α as biomarkers of rejection. The analysis revealed that GLuc significantly outperformed Attorney Docket No. 29539-0832WO1 / MGH 2024-313 TNF-α, with an area under the curve (AUC) exceeding 95%, indicating its superior reliability in detecting rejection (FIG. 11F). Finally, given that transplanted patients typically undergo immunosuppressive regimens, we aimed to explore how our biosensor would respond in these scenarios. Engineered cells were treated with increasing doses of Cyclosporine A (CsA), a widely used immunosuppressive agent for managing organ rejection post- transplantation, and subsequently activated with TNF-α. Biosensor activation was assessed via supernatant GLuc secretion, revealing a clear dose-response to immunosuppression with significantly lower GLuc levels (FIG. 11G). Notably, even the lowest dose of CsA effectively inhibited biosensor activation, resulting in significantly lower GLuc secretion compared to the positive control group, that received only TNF-α. This further highlighted the sensitivity of this genetic biosensor platform. Example 9: Genetically engineered smart organs enabled early in vivo detection of rejection The inducible genetic construct was ultimately validated in genetically engineered VCAs for its ability to detect rejection. Organs were perfused with lentiviral vectors expressing the construct of interest using our optimized perfusion protocol, then transplanted in a partial mismatch orthotopic transplant model and monitored for 11 days (FIGS. 18A-18F). Control groups consisted of animals that received no mismatch transplants, and, therefore, did not undergo rejection, as well as animals that received mismatch transplants but were administered daily doses of the immunosuppressive agent CsA from POD 2 onwards. Clinical evolution showed erythema and indurated tissue in rejection VCAs from around POD 5 onwards, increasing in severity (FIG. 18B). Non-rejection animals showed normal aspect of the skin, no induration, and hair growth by the end of the first week. Rejection under immunosuppression (IS) animals exhibited limited erythema. Tissue analysis confirmed transgene expression in rejected VCA tissues, showing the highest levels in the vasculature, consistent with previous observations (FIG. 18C). In vivo bioluminescent imaging revealed a more than 10-fold higher signal in rejected VCA at POD 4 (FIG. 12A). Accordingly, plasma GLuc levels were significantly higher in rejection animals compared to both the non-rejection and Attorney Docket No. 29539-0832WO1 / MGH 2024-313 rejection under IS group (FIG. 12B). Remarkably, while GLuc peaked at POD 3 in rejection animals, histological analysis revealed acute cell-mediated rejection as late as between PODs 9 and 11 (FIGS. 18D, 18E). Off-target organs (heart, lung, liver, spleen, kidney, intestine, contralateral skin, contralateral muscle, contralateral femoral vessels) exhibited no histological evidence of pathological changes (FIGS. 19A-19I). Plasma cytokine secretion was also monitored postoperatively and compared between rejection and non-rejection animals (FIG. 18F). When evaluating GLuc, Banff score, and several pro-inflammatory cytokines as rejection biomarkers through ROC analysis, GLuc demonstrated a significantly superior performance, achieving an AUC of over 90%, well above the other candidates. (FIG. 12C). FIGS. 12D-12I present a detailed ROC analysis for each biomarker. Notably, none of the examinedcytokines—selected for their relevance in VCA rejection38,39—outperformed GLuc asa rejection biomarker. This aligned with our expectations, given that these cytokines were not as specific to local rejection events, and our results indicated they were also less sensitive. Accordingly, when assessing the time-to-diagnose probability for each biomarker, GLuc again demonstrated superior performance, enabling the earliest detection and diagnosis of rejection compared to the other evaluated candidates (FIG. 12J). These findings collectively highlighted the robustness of our genetically engineered biosensor platform as a reliable method for early rejection detection in VCA, highlighting its specificity and sensitivity compared to traditional and alternative markers. This positioned our biosensor as a promising tool for improving clinical outcomes through timely intervention in rejection scenarios. Example 10: Anti-IL-6 gene therapeutics delayed the onset of VCA graft rejection. IL-6 signaling plays a pivotal role in the orchestration of organ rejection (Ma et al., Front Immunol. 2021 Oct 28;12:778359). Soluble GP130 (sGP130) specifically inhibits IL-6 trans-signaling (Garbers et al., J Biol Chem. 2011 Dec 16;286(50):42959-70). The pLV-EF1a-GLuc-IRES-mRFP plasmid was modified to expresses both the biomarker Gluc and sGP130 (hIL6ST[ORF024283]) under the constitutive EF1α promoter (see vector map at FIG. 21). Lentiviral vectors and lentivirus for in vivo use was prepared as described (see Materials and Methods). Rat Attorney Docket No. 29539-0832WO1 / MGH 2024-313 hindlimbs (i.e., model VCAs) were procured and transplanted as described. Normothermic machine perfusion parameters were as follows: Normothermic Perfusion: 37oC; Perfusate: Modified Steen+;Total perfusion time: 6 h; Virus injection at 30 min; Virus flush-out at 5:30 min; and Flow rate: 1.5 mL / min. Histological analysis revealed a delayed onset of rejection – as late as POD 14 – in animals transplanted with VCA comprising the Gluc and sGP130 virus (FIG. 22). Animals with the genetically modified VCA also demonstrated delayed erythema in the transplanted hindlimbs (FIG. 23). References 1 United Network for Organ Sharing. 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Relation between stress and cytokine responses in inner-city mothers. Ann Allergy Asthma Immunol.2015;115:439–45.e3. OTHER EMBODIMENTS It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No. 29539-0832WO1 / MGH 2024-313 WHAT IS CLAIMED IS:

1. A method of making a genetically modified organ, the method comprising ex vivo perfusion of a donor organ with an effective amount of a viral vector for transduction of an immunosensor transgene into a genome of at least one cell of the donor organ.

2. The method of claim 1, wherein the viral vector comprises a nucleic acid sequence encoding the immunosensor transgene, a promoter for expression of the immunosensor transgene, and a response element that directs expression of the immunosensor transgene in response to a physiological stimulus, optionally wherein the physiological stimulus is inflammation.

3. The method of claim 1 or 2, wherein the immunosensor transgene expresses a secretable biomarker, optionally wherein the secretable biomarker is Gaussia Luciferase (GLuc).

4. The method of any one of claims 1-3, wherein the response element is from nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB).

5. The method of any one of claims 1-4, wherein the viral vector is selected from the group consisting of retroviruses, adenoviruses, oncoretroviruses, lentiviruses, spumaviruses, adeno-associated viruses, and herpes simplex viruses, preferably wherein the viral vector is a lentivirus.

6. The method of any one of claims 1-5, wherein the viral vector comprises a nucleic sequence at least 95% identical to SEQ ID NO:

1.

7. The method of any one of claims 1-6, wherein the viral vector further comprises a nucleic acid sequence encoding a therapeutic protein, optionally wherein the therapeutic protein comprises soluble GP130 (sGP130).Attorney Docket No. 29539-0832WO1 / MGH 2024-313 8. The method of claim 6, wherein the therapeutic protein is fused to the secretable biomarker.

9. The method of any one of claims 1-8, wherein the genetically modified organ is a solid organ.

10. The method of claim 9, wherein the solid organ is selected from the group consisting of kidney, liver, heart, lung, pancreas, and intestine.

11. The method of any one of claims 1-8, wherein the genetically modified organ is a vascularized composite allograft.

12. The method of claim 11, wherein the vascularized composite allograft is selected from the group consisting of face, limb, long bones soft tissues, uterus, bladder, abdominal wall, musculoskeletal composite graft segments, penis, adrenal glands and thymus glands.

13. A method of making a genetically modified organ, the method comprising: (a) harvesting an organ from a donor; (b) placing the organ in a reservoir of a perfusion machine; (c) submerging the organ / tissue with a perfusion solution; (d) injecting the viral vector comprising a nucleic acid sequence encoding a secretable biomarker, a promoter for expression of the secretable biomarker, and a response element that directs expression of the secretable biomarker; (e) circulating the perfusate comprising the viral vector in the reservoir of the perfusion machine via closed circuit circulation; and (f) flushing the perfusate comprising the viral vector out of the organ.

14. The method of claim 13, wherein the organ is submerged in the perfusion solution for at least about 30 minutes before injecting the viral vector.

15. The method of claim 13 or claim 14, wherein steps (c)-(f) occur at about 37°C.Attorney Docket No. 29539-0832WO1 / MGH 2024-313 16. The method of any one of claims 13-15, wherein the perfusate comprising the viral vector is circulated in the reservoir of the perfusion machine for about 5 to 12 hours.

17. A method of monitoring post-transplantation surgical outcome in a subject who has had an organ transplant, the method comprising: transplanting a genetically modified organ into the subject, wherein the genetically modified organ was prepared according to any one of claims 1-19; collecting a biological sample from the subject after transplantation of the genetically modified organ; and measuring the amount of secretable biomarker in the biological sample.

18. The method of claim 17, the method further comprising administering at least one immunosuppressant treatment to the subject to prevent or delay the onset of organ rejection if the secretable biomarker is detected in the biological sample.

19. The method of claim 17 or claim 18, wherein the secretable biomarker is Gaussia Luciferase (GLuc).

20. The method of claim 19, wherein GLuc is measured in the biological sample by a method selected from the group consisting of a GLuc substrate assay, ELISA, Western blot, and qRT-PCR.

21. A genetically modified organ comprising at least one immunosensor transgene, wherein the immunosensor transgene expresses a secretable biomarker in response to a physiological stimulus.

22. The genetically modified organ of claim 21, wherein the secretable biomarker is Gaussia Luciferase (GLuc).Attorney Docket No. 29539-0832WO1 / MGH 2024-313 23. The genetically modified organ of claim 21 or claim 22, wherein the immunosensor transgene further expresses a therapeutic protein in response to a physiological stimulus.

24. The genetically modified organ of any one of claims 21-23, wherein the therapeutic protein comprises soluble GP130 (sGP130).

25. The genetically modified organ of any one of claims 21-24, wherein the physiological stimulus is inflammation.

26. A viral vector comprising a nucleic acid sequence encoding an immunosensor transgene, a promoter for expression of the immunosensor transgene, and a response element that directs expression of the immunosensor transgene in response to a physiological stimulus.

27. The viral vector of claim 26, wherein the viral vector comprises a nucleic sequence at least 95% identical to SEQ ID NO:

1.

28. A genetically modified organ comprising at least one immunosensor transgene, wherein the genetically modified organ is transduced with the viral vector of claim 26 or claim 27.

29. A genetically modified organ, wherein the genetically modified organ is prepared according to the methods of any one of claims 1-19.

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