Reframing Technology and Ethics in Corneal Xenotransplantation: A Scoping Review of Standards and Safety in Ophthalmology

ABSTRACT

Xenotransplantation offers a potential solution to the critical global organ shortage by allowing the transplantation of organs from non‐primate animals to humans. To address the critical barriers of biological safety, zoonotic infections, and immunological rejection, this review evaluates current innovations in genetically engineered donor species strategies and assesses existing ophthalmological standards, employing specific expression of human regulatory proteins (hCD46/hCD55) and genetically modified triple knockout (GGTA1/B4GalNT2/CMAH), biophysical thresholds, porcine cytomegalovirus screening, bacterial/mycotic cultures, and DNA sequencing for unidentified organisms. Employing a snowball strategy across PubMed Central, ResearchGate, and Wiley Online Library, 47 articles were selected and reported following the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses Extension for Scoping Reviews. The feasibility of xenotransplantation relies heavily on advancements in donor technology and genotype transparency, animal husbandry and biosafety, infectious testing and surveillance, recipient monitoring and lifetime surveillance, biosafety incident and response plan, data governance, sharing and oversight, and animal welfare and social license, necessitating pathogen observation, biosecure storage, bioexclusion monitoring, and good manufacturing practice‐style manual, quantitative polymerase chain reaction for porcine cytomegalovirus, porcine reproductive and respiratory syndrome virus, hepatitis E virus, alongside porcine endogenous retrovirus‐A/B/C; metagenomic sequencing initially, followed by 1, 3, 6, and 12 months, and subsequently once every year, systematic and preliminary persistent sampling evaluations, independent data safety monitoring board, collaborative guidelines, encrypted databases, and data retention. The integration of endothelium‐protective human genes, including hA20 or hHO‐1, into genetically altered donor corneas in non‐human primate xenografts, is expected to minimize initial post‐transplant endothelial cell density loss by ≥ 20% compared to existing models. While clustered regularly interspaced short palindromic repeats‐based modifications offer transformative potential for xenotransplantation, their reliability currently depends on establishing stringent biosafety standards.

Bringing attention to the moral dilemmas raised by xenotransplantation and the continuous creation of standardized frameworks for donor technology and biosafety risk management, with a focus on striking a balance between patient safety, medical innovation, and ethical obligation.

Abbreviations

CRISPR

clustered regularly interspaced short palindromic repeats

NHP

non‐human primate

PCMV

porcine cytomegalovirus

PCR

polymerase chain reaction

PERV

porcine endogenous retrovirus

1. Background

Xenotransplantation describes the transplantation of living tissues, or organs, from one species into another, most frequently transferring them from animals to humans. Although specific animal‐delivered tissues like pig heart valves have been effectively utilized for a long time—driven by their biological inertness and minimal immunogenicity—recent studies are investigating the transplantation of functional tissues, including corneas, to resolve acute constraints. Such an approach has garnered significant consideration, specifically in employing pig corneas to confront the worldwide depletion of biological tissue donations, mostly in developing regions like Asia. The scope of xenotransplantation includes corneal graft transplantation to reconstruct visual function and substitute for life‐saving organs, despite simultaneously raising significant concerns about immunologic rejection and zoonotic transmission of disease [1, 2, 3, 4]. The advantages of employing xenogeneic tissue over fabricated scaffolds include a strong resemblance in chemical profile and micro‐architecture to the human cornea, ready availability, lower cost, and identical optical and biomechanical attributes comparable to those of the natural cornea [5, 6].

Allogeneic rejection remains the most prevalent cause of corneal graft failure, irrespective of the cornea's immunological privilege and its distinct constraints, as illustrated in Figures 1 and 2. For first‐time recipients without corneal vascularization, 2‐year survival rates are around 90%, but in high‐risk instances, it declines to 35%. One‐third of grafts manifest immune‐mediated rejection indications, which interfere with corneal transparency and cause the loss of donor endothelial cells [8].

Figure 1.

Corneal graft survival and rejection risk. Note: This figure is compiled by the authors based on data drawn from previously published studies [2, 3, 4].

Figure 2.

Bar chart showing the distribution of causes of corneal graft failure based on a study of 638 graft failures in India [7]. This figure is compiled by the authors based on data drawn from previously published studies [3, 4, 6]. IOP, intraocular pressure.

Corneal opacity, one of the top five causes of blindness globally, is thought to be responsible for 3.2% of all cases of blindness and 1.3% of all instances of moderate to severe visual impairment. Over 12.7 million individuals are thought to be on the waiting list for corneal transplants worldwide, with the shortage being especially severe in low‐ and middle‐income countries where eye banking facilities are frequently nonexistent. Only one donor cornea is available for every 70 needed globally, according to recent studies that show a startling difference in availability [1, 9]. Early in the 17th century, xenotransplantation was performed, with the earliest studies centering on the transplantation of organs from primates. Non‐primate species, including goats, pigs, and rabbits, were also used in experiments, despite varying levels of efficacy. Technological innovation has made it possible to effectively transplant organs obtained from non‐primate animals into humans. Whereas non‐human primates, for instance, gibbons, have previously been adopted as donor grafts, their elevated probability of infection and substantial reproductive cost burden render them unsuitable for clinical application. The deliberate choice of species for corneal tissue procurement depends on variables such as protein structural analogy to the human cornea, anatomical dimensions, accessibility, and effectiveness. Despite scientific probing into the protein profile of the human cornea, evidence on the distinctions in the content and arrangement of corneal proteins between human beings and various species is lacking [1, 5, 10, 11]. Over 1.3 million people in India are experiencing bilateral corneal blindness, ranking it as the second most prevalent and preventable cause of blindness. Considering the country has nearly 10 million fatalities annually, about 55,260 corneas were obtained in 2019, and only 26,416 were utilized. The predicament intensified due to the COVID‐19 outbreak, which prompted an abrupt decline in tissue procurement to 21,493, with only 13,323 transplants in 2020. India must aim to acquire at least 200,000 donor corneas annually to adequately meet the country's requirement and eliminate the backlog, assuming the existing corneal tissue application rate is around 60% [12]. An accumulation of instances of corneal blindness results from an extensive shortage of donor corneas driven by insufficient donations, inefficient tissue utilization, and inadequate equipment. This illustrates the urgent need to enhance the eye banking and cornea transplantation framework through effective scheduling, allocation of resources, and education among the public [12]. Porcine corneas are an extremely effective and reliable donor specimen for translational studies due to their physiological and anatomical similarity to human corneas, genetic adaptability (e.g., clustered regularly interspaced short palindromic repeats [CRISPR] modification minimizes immunogenicity), scalability, and biocompatibility [3, 5]. Xenotransplantation has the potential to resolve the significant tissue shortage by exhibiting genetically altered pig tissues as suitable alternatives. Employing cellular and whole organ transplants, it has shown potential in treating medical conditions involving diabetes, Parkinson's disease, and organ dysfunction. Research continues to advance rapidly since pigs have become easier to raise and are more ethically acceptable than primates. Yet, potential biological safety, zoonotic infections, and immunological rejection remain significant concerns regarding its clinical application and governance [4].

2. Methods

2.1. Scoping

This scoping review aims to map the existing literature on technology and ethics in corneal xenotransplantation and to identify current standards and safety protocols in ophthalmology.

2.2. Planning

Journal platforms such as PubMed Central, ResearchGate, and Wiley Online Library were searched. In regard to the study's relevance, the literature review concentrated on keywords and Medical Subject Headings corresponding to “Corneal Transplantation/methods,” “biomechanics,” “immune response,” “Graft Rejection/prevention & control,” “Graft Survival,” “genetically‐engineered,” “Corneal Transplantation,” “Xenotransplantation,” “Corneal Xenotransplantation,” “Swine,” “Animals,” “India/epidemiology,” “Transplantation, Heterologous*/methods,” “Transplantation, Heterologous/methods,” “Transplantation, Heterologous/trends.”

2.3. Identification

Employing the iterative snowballing strategy that systematically examined both the reference lists (backward snowballing) and citing articles (forward snowballing) of an initial set of articles, the researchers identified, screened, and included relevant articles by evaluating references and citations in key studies. The Preferred Reporting Items for Systematic Reviews and Meta‐Analyses Extension for Scoping Reviews flow diagram outlines this method. Subsequent to the removal of 14 duplicates, 47 articles were selected following a methodical screening procedure. Ten studies were deemed ineligible in the full‐text evaluation due to contextual insignificance (six studies) and inadequate information (four), while five full‐text papers were not retrievable, even with extensive literature retrieval efforts and attempts to contact the authors (Figure 3).

Figure 3.

Preferred reporting items for systematic reviews and meta‐analyses extension for scoping reviews flow diagram.

3. Results

3.1. Overview of Current Animal‐to‐Human Transplantation Attempts

In the past few decades, xenotransplantation has transformed considerably, and currently, one of the most extensively studied therapeutic avenues is corneal xenografts. Innovative developments in decellularization approaches, which endeavor to strip away antigenic cellular elements while preserving the extracellular matrix, have revived interest in corneal transplanted xenogeneic grafts. Porcine corneas remain physiologically and anatomically compatible with human tissue among potential donor species, especially with respect to collagen content and optical appearance, making them suitable prospects for medical translation. Yet concerns persist, especially with xenoantigens including α‐Gal and NeuGc, remnant antigenicity, and zoonotic transmission risks. However, immunomodulatory modalities and genetic engineering‐based inventions comprising α‐Gal mutant specimens have shown some preliminary responses; additional research must be conducted to validate long‐term transplantation survivability and physiological incorporation in recipients. Porcine tissue‐derived xenogeneic heart valves are currently in extensive clinical use, suggesting comparable approaches could be implemented on ocular tissues [5, 9, 10, 11].

3.2. Documented Ethical Challenges

Clinical studies employing xenotransplantation raise concerns about the efficacy of safety measures and social assurance. The therapeutic misconception is a frequent concern, specifically for critically ill individuals who lack alternative treatment regimens. Because these participants are motivated to contribute, the credibility of informed consent is impaired by their vulnerability, which causes them to inaccurately presume that innovative therapies are therapeutic rather than experimental. Considering that such marginalization not only sustains disparity but also implies a systemic incompetence of confronting discontent founded on previous incidents, it impacts ethical concerns regarding clinical application. Furthermore, as participants will not be effectively abandoned despite disrupting public health regulations, prerequisites for lifelong follow‐up—and potentially supervision of recipient personal interaction—would challenge the research's theory of voluntary behavior. Subsequently, a significant accountability vacuum has recently been highlighted because authoritative institutions have yet to offer sufficient information concerning how activities for transplant surveillance, instruction, and conformity ought to be allocated or imposed. Such persistent conflicts corroborate that xenotransplantation endeavors cannot be implemented ethically without a comprehensive and transparent ethical framework that supersedes the existing fragmented strategies [13].

3.3. Biosafety Risks: Zoonotic, Genetic Instability

Biosafety concerns related to inheritance instability and zoonotic dissemination are evidenced by preliminary research and prospective clinical xenotransplantation initiatives. Porcine cytomegalovirus/porcine roseolovirus has significantly impacted transplant outcomes. For instance, in the initial porcine‐to‐human heart transplantation project, it precipitated graft rejection and patient mortality, and in baboon studies, it reduced graft longevity. Especially in immunocompromised recipients, infection with hepatitis E virus genotype 3 constitutes a potential zoonotic complication. The pig genome encodes 40–60 copies of porcine endogenous retroviruses (PERVs), which can infect human cells in vitro but have not yet been observed to replicate in clinical or research instances. Additionally, hereditary susceptibility aggravates these potential hazards: while CRISPR/Cas9‐induced mutations have successfully inhibited PERVs and limited immunogenicity, they also introduce the risk of chromosomal alterations and off‐target modifications, which may undermine graft reliability. Standardized biosafety appraisals are complicated by the observation that proviral copy numbers vary within organs, implying persistent retro‐element activity. To enhance clinical safety, these outcomes cumulatively establish that inheritance instability and zoonotic dissemination remain substantial constraints, recommending genomic integrity screening, pathogen‐free cultivation, and extended virological surveillance. One recommended standardized protocol for xenotransplantation health is a multilayered biosafety strategy comprising full serological and microbiological screening, droplet digital polymerase chain reaction (PCR) for PERV detection, and management of certified pathogen‐free donor herds. Longitudinal voluntary nasal swabs with evaluation of viral transmission period and cycle threshold‐symptom correlations improve clinical bio‐surveillance and reinforce translational risk identification, according to associated human surveillance information from solid organ transplant recipients [14]. To provide repeatable, regulatory‐aligned surveillance, this result should be combined with standardized PCR‐based, immunological, and validated screening technologies, as well as longitudinal herd surveillance and established pathogen‐free colony protocols, to increase biosafety evaluation [15, 16].

3.4. Overview of Donor Animal Genetic Modification Technologies

To conquer immunological restrictions in xenotransplantation, current innovations in genetically engineered donor species strategies are significant. Porcine models remain the recommended donors because they are affordable to reproduce, present fewer ethical questions, and are more adaptive for DNA modifications as compared to non‐human primates employed in initial research. A significant accomplishment occurred in the early 2000s when α−1,3‐galactosyltransferase gene‐knockout porcine models became available, which minimized hyper‐acute rejection. To lessen the rejection rate, over 40 genetic alterations were recently executed that incorporate human genes responsible for the complement cascade, coagulation pathways, and immune and inflammatory responses. By facilitating simultaneous gene modification and eliminating epitopes associated with antigenicity and PERVs, the CRISPR‐Cas9 approach significantly accelerated the method, resolving rejection along with zoonotic complications. Overall, these strategies have made genetically modified porcine cells accessible for transplant resources in therapeutic contexts [17, 18]. Such innovations enable distinctive biological donors to exhibit lower antigenicity; yet, these tissues require an effective risk estimation framework that integrates proteomic parameters like additive proteins and NF‐κB1 networks for predicting graft responses. Structured biosafety is also imperative for ensuring genetic integrity, reducing zoonotic dissemination, and facilitating the secure clinical application of xenografts [19]. Proteomic investigation of cardiac xenografts in a pig‐to‐non‐human primate paradigm revealed considerable overexpression of proteins, including PTX3, MVP, and HSP90AB1, among all the groups being studied. These proteins, which utilize the NF‐κB signaling pathway as a downstream regulator, underscore a pivotal pathway of rejection. Inhibition of NF‐κB with BAY 11‐7082 decreased inflammation and extended graft life, confirming its viability as a potential therapeutic approach [14, 15, 16, 20].

4. Discussion

4.1. Translational Perspectives in Corneal Xenotransplantation

4.1.1. Innate Immunological Responses and Graft Rejection

Corneal xenograft rejection is acutely mediated by multi‐modal innate immune dysregulation [21]. At the biomolecular level, rejection is driven by interspecies incompatibilities—specifically the attenuation of the porcine CD47‐human SIRPα inhibitory axis—which precipitates macrophage activation and subsequent phagocytic activity. Concurrently, the pro‐inflammatory environment is compounded by neutrophil extracellular traps, which propagate host‐mediated tissue degradation [22]. Persistent graft survival is compromised by residual matrix antigens, despite decellularization effectively minimizing baseline immunogenicity [3]. Clinically, regardless of therapeutic intervention, standard immunosuppression is insufficient to prevent intense, antibody‐independent innate cascades, leading to resistant postoperative morbidities (edema, opacity, and neovascularization). The precise biomolecular pathways that correlate this acute innate reaction to chronic transplant rejection remain inadequately characterized [23].

4.1.2. Biomechanical Compatibility and Tissue Integration

While porcine corneas approximate human refractive compatibility, they diverge considerably in morphology and viscoelasticity, as shown in Table 1. Characterized by a thicker stroma and epithelium (7–9 layers), porcine tissue exhibits lower rigidity, lower stiffness, and higher viscoelastic creep compared to human corneas. Consequently, these variables significantly impact post‐surgical shape retention and refractive predictability in xenotransplantation [1, 24, 25].

Table 1.

Comparison of morphological and biomechanical comparison of human and porcine corneas.

Aspect	Human	Porcine	References
Epithelium layers	5	7–9	[1]
Stromal thickness	Baseline	30% thicker in pig	[24]
Overall stiffness	Higher	Lower, more viscoelastic	[25]

The clinical efficacy of porcine corneal xenografts is significantly impeded by intrinsic structural and cellular discrepancies. The inherent thickness and diminished tensile stiffness of porcine corneas prevent sustained shape retention and stability, inducing a biomechanical degradation that negatively impacts refractive accuracy and overall visual performance, as described in Figure 4 [26, 27, 28].

Figure 4.

Biomechanical discrepancies and postoperative consequences. This figure is compiled by the authors.

To reduce the biomechanical and immunological consequences of porcine xenografts, various advanced scientific and engineering methods are actively employed. First, genetic engineering of donor corneas overcomes fundamental immunological challenges by modifying carbohydrate antigens and enhancing endothelial stability. Additionally, decellularization and recellularization eliminate immunogenic porcine cells and replace the cellular matrix with human cells, restoring natural barrier activities and biomechanical stability. To reduce structural inadequacies, ex vivo mechanical conditioning—involving a combination of compression, gamma irradiation, and substantial cross‐linking—enhances stromal rigidity to inhibit viscoelastic creep. Furthermore, corneal tissue bioengineering employs purified porcine collagen (through intensive extraction of cells, DNA/RNA, and xenoantigens) to create stable, cell‐free scaffolds that efficiently correspond to human mechanical strength and effectively restore refractive acuity [29, 30, 31, 32]. While these bioengineering approaches effectively address structural and immunological barriers, effective neural regeneration and functional restoration are crucial for the clinical success of porcine xenografts in order to restore corneal homeostasis fully.

4.1.3. Neural Regeneration and Functional Recovery

Corneal nerves are essential for preserving corneal sensitivity, epithelial integrity, metabolism, and ocular surface homeostasis. Post‐corneal xenotransplantation, the restoration of the neural network poses a considerable challenge, potentially leading to diminished corneal sensitivity, prolonged epithelial recovery, and postoperative discomfort compared with allogeneic grafts [33].

As delineated in Figure 5, the timeline of corneal neural re‐innervation follows a triphasic pattern. The initial stage (Days 3–20) is characterized by the rapid infiltration of host‐derived nerve sprouts into the donor cornea graft. In vivo monitoring validates that this host‐nerve regeneration is identifiable as early as Day 3, advances to the paracentral cornea by Day 7, and attains the peripheral cornea by Day 20. This acute onset of infiltration correlates with the total degeneration of the donor corneal nerves, which tend to disappear by Day 3. Subsequently, the mid‐term stage (3–6 months), which is marked by the architectural regrowth and sensation of the sub‐basal nerve plexus, facilitates a partial restoration of corneal sensitivity. Ultimately, throughout the long‐term phase (12–24 months), nerve density and functional sensory thresholds stabilize; despite this, denser stromal nerves often exhibit structural aberrations compared to healthy, un‐operated controls [34, 35, 36].

Figure 5.

Time frame of corneal re‐innervation after grafting. This figure is compiled by the authors.

4.2. Strategic Pathway for Inter‐Species Eye Tissue Transfer to Enhance Compatibility With Xenotransplantation

Table 2 specifies relevant experimental variables for studies involving xenotransplantation. It assures an extensive assessment and safety of porcine corneal grafts by incorporating genetic modifications for compatibility, biophysical thresholds for corneal health, validated preservation protocols, comprehensive immunosuppression strategies, detailed preclinical outcome metrics, and stringent infectious safety screening, involving PERV and PCMV detection.

Table 2.

A strategic framework for eye tissue compatibility for inter‐species eye tissue.

Number	Parameters	Experimental measures	References
1	Genotype	Employing specific expression of human regulatory proteins (hCD46/hCD55) and genetically modified triple knockout (GGTA1/B4GalNT2/CMAH)	[37, 38, 39]
2	Biophysical threshold	Transparency (slit‐lamp grade) upon removal, endothelial cell count at baseline (endothelial cell density: 2500/mm2), and corneal thickness (central corneal thickness: 600 μm) should exhibit satisfactory enhancement subsequent to 7 days in short‐term measurement	[37, 39, 40]
3	Preservation standards	The preservation temperature, description of the solution with terminologies, and endothelial survival evaluation technique must be documented, and certified corneal preservation method, such as Optisol‐GS or an equivalent solvent, must be employed having a specified permitted cold ischemia time of 7 days	[37, 38, 39, 40]
4	Preclinical measures	Graft transparency, endothelial cell density alteration, and morphology are instances of primary outcomes; histology, donor‐specific antibody profile, complement activation biomarkers, and pathogenic screening are evidence of secondary endpoints	[37, 38, 39, 40]
5	Immunosuppression	The guideline calls for identifying the topical regimens, medications for maintenance (such as specified calcineurin inhibitors and costimulation inhibition remedies with details concerning therapeutic drug surveillance), topical dosage (drug, dosage, and route), and an extensive rationale for each determination	[37, 38, 39, 40]
6	Infectious safety and PERV screening	PCMV screening, bacterial/mycotic cultures, DNA sequencing for unidentified organisms, and qPCR for PERV‐A, B, and C have been components of the entirety of the pathogen screening array. All positive and negative outcomes ought to be reported, in addition to the precise methodologies that were employed	[37, 38, 39, 40]

Abbreviations: DNA, deoxyribonucleic acid; PCMV, porcine cytomegalovirus; PERV, porcine endogenous retrovirus; qPCR, quantitative polymerase chain reaction.

4.3. Ethical‐Biosafety Minimum Standards Instrumentation

As mentioned previously, Table 3 highlights the proposed study objective regarding Ethical‐Biosafety Minimum Standards framework, which incorporates the following provisions: provenance and donor authenticity, unbiased validation of apparent donor‐genotype evidence; biosecure husbandry concerning traceability to inhibit zoonotic organisms from donor resource groups from establishing themselves and proliferating, a prerequisite for assessment (quantitative polymerase chain reaction for identified agents + metagenomic sequencing for unidentified pathogens), a lifetime recipient surveillance plan to maintain accountability and preserve confidential clinical information, and openly accessible informed‐consent guidelines that explicitly acknowledge a possible PERV risk and the necessity for persistent follow‐up [37, 38, 39, 40, 41]. The scarcity of human contributors for corneal transplantation can be overcome through methods involving genetic modifications, including cloning and CRISPR. Porcine models exhibit extended graft survival in transgenic pigs due to reduced immunological rejection. Despite the potential offered by precise gene alteration, additional procedures incorporating broad immunosuppression are still required for enduring prolonged and constant xenocorneal transplantation, as evidenced by the lack of substantial prognostic advancement after CRISPR‐associated multi‐gene transformations [42, 43].

Table 3.

Ethical‐biosafety standards instrumentation.

Number	Parameter	Biomarker	Threshold	Rationale	References
1	Donor technology and genotype transparency	Human transgenes; PERV suppression status; GGTA1, CMAH, B4GALNT2 knockouts; validated genotype sequencing	Mandatory documentation of genetic modifications, reproductive records, and sequencing validation	Assures provenance and donor authenticity regarding biosafety.	[40, 41, 42]
2	Animal husbandry and biosafety	Pathogen monitoring; biosecure storage; bioexclusion systems; GMP‐style manual	Continuous pathogen surveillance and controlled biosecure breeding conditions	Inhibits zoonotic organisms from donor resource groups from establishing themselves and proliferating	[41, 42, 43]
3	Infectious testing and surveillance	qPCR for PCMV, PRRSV, HEV, PERV‐A/B/C; metagenomic sequencing	Pre‐transplant screening; follow‐up at 1, 3, 6, 12 months, then annually	Mitigates zoonotic risk by screening both identified and unidentified infectious pathogens	[40, 42]
4	Recipient monitoring and lifetime surveillance	Systematic sampling (blood, urine, ocular surface)	Persistent and lifelong surveillance authorization with periodic evaluations	Considers sustainability and ethical accountability regarding public health	[40, 44]
5	Informed consent—minimum elements	Perpetual supervision standards; public health documentation; infectious risk disclosure (e.g., PERV)	Mandatory inclusion of long‐term monitoring and infectious consequence disclosure	Makes certain that xenotransplant clinical trials are transparent and accessible	[43, 44]
6	Biosafety incident and response plan	Xenozoonosis indicators; contact tracing; isolation protocol; prophylactic regimen	Immediate reporting and containment protocol framework	Stipulates an immediate approach to prevent prospective zoonotic outbreaks and report them to authorities	[40, 45]
7	Data governance, sharing and oversight	Independent DSMB; encrypted databases; data retention policy	Defined oversight board and secure data‐sharing guidelines	Maintains accountability and preserves confidential clinical information	[40, 41, 44]
8	Animal welfare and social license	Community transparency; ethical inspections; stakeholder engagement	Compliance with welfare regulations and socio‐ethical standards	Preserves xenotransplantation legally feasible and socially acceptable	[41]

Abbreviations: DSMB, data safety monitoring board; GMP, good manufacturing practice; HEV, hepatitis E virus; PCMV, porcine cytomegalovirus; PERV, porcine endogenous retrovirus; PRRSV, porcine reproductive and respiratory syndrome virus; qPCR, quantitative polymerase chain reaction.

In principle, donor innovations derived from CRISPR and transgene expression exhibit potential in enhancing corneal xenotransplantation, but their efficacy is inconsistent. Graft survival can be prolonged by regional expression of immunomodulatory biomarkers like hCTLA4‐Ig, even as humoral aspects of broader immune responses continue to raise concerns. Consequently, when combined with additional immunosuppression approaches, genetically modified donated corneas could serve as an effective substitute for a human corneal transplant [42]. When summarized, CRISPR‐based modification and cloning present promising donor innovations for xenotransplantation; however, their effective implementation necessitates stringent biosafety standards and extensive risk evaluation [44]. For donor animal methodologies, such as genetic modification and decellularization, to be regarded as secure, accessible, and reliable, internationally accepted norms must be implemented [4, 13]. For sustainable clinical translation, an effective ethical and statutory framework can be reinforced by multidisciplinary collaboration, providing comparable significance to patient satisfaction and biosafety risk reduction.

5. Conclusions

An intricate equilibrium between scientific innovation and societal accountability must be maintained in order to resolve the ethical concerns pertaining to xenotransplantation. Porcine corneal xenotransplantation is an achievable and remedial means for the scarcity of human donor corneas. Nevertheless, it also introduces complications associated with mutations in genes, graft adverse reactions, zoonotic propagation, and longitudinal biosafety [3, 9]. A spectrum of ethical concerns relevant to xenotransplantation clinical trials must be addressed before responsibly initiating human research. Patient rights and public health mandates must be effectively considered, as evidenced by instances including therapeutic misconception, equitable access and accountability, and the ethical consequences of long‐term investigation. Furthermore, the prospective necessity to broaden supervision and knowledge activities involves barriers that existing legislative regimes do not sufficiently address. The aforementioned constraints underscore the necessity for rigorous ethical standards, transparent governance, and substantial community engagement to ensure that therapeutic progression aligns with both human well‐being and social assistance, without compromising the scientific clinical potential of xenotransplantation [13].

Data in Table 4 suggest advancements have been made in evaluating short‐run biophysical exposure compatibility between genetically altered porcine donor cells and in‐vitro micro‐vascular (endothelium) evaluation. Evidently, substantial gaps remain; for instance, there is inadequate availability of comprehensive standardized preservation protocols, explicit parameter reporting, and consistent non‐human primate evidence demonstrating prolonged survival of endothelial cells. Prospective studies are urgently required to evaluate the potency of immunomodulatory transcripts to determine the translational accuracy, reliability, and prospective cell composition of donors. Despite corneal xenotransplantation exhibiting promising graft survival and transparency in preclinical and preliminary clinical research, issues concerning neural regeneration, prospective graft stability, and persisting functional impacts remain largely unexplored. While some studies emphasize the potential risk of graft degradation and late rejection, others establish good biocompatibility and immunological tolerance.

Table 4.

The translational objectives and unsolved scientific needs for prospective corneal xenotransplantation initiatives.

Donor genotype	Model	Primary outcomes	Associated research gap
Triple Knockout (GGTA1−/−, CMAH−/−, B4GALNT2−/−) + human complement regulators (CD46, CD55)	(1) Ex vivo human cornea interface (2) Short‐term in vitro assays (3) NHP trials planned	(1) Maintenance of endothelial viability and transparency (> 90% clarity at 7 days) (2) ECD loss (< 10%)	(1) Lack of long‐term (≥ 6 months) NHP survival data (2) Constrained insight regarding endothelial immune adaptation
Double Knockout (GGTA1−/−, CMAH−/−)	Ex vivo porcine‐to‐human (cold storage model)	(1) Comparable biophysical and optical properties to human corneas (2) Endothelial morphology preserved for 7 days	(1) Small sample size (2) Absence of immune parameter analysis
Quadruple Knockout (GGTA1−/−, CMAH−/−, B4GALNT2−/−, PERV−/−)	(1) In vitro bio‐compatibility assays (2) Early‐stage NHP validation	(1) Improved complement resistance (2) Higher endothelial stability	(1) Requires validation of PERV inactivation under physiological stress (2) In vivo graft conditions
Wild‐type porcine corneas (controls)	Ex vivo comparison with human donor corneas	(1) Rapid endothelial cell loss (2) High immune reactivity within 48–72 h	Mechanism of rapid rejection incompletely understood

Abbreviations: ECD, endothelial cell density; NHP, non‐human primate; PERV, porcine endogenous retrovirus.

Two plausible hypotheses may facilitate subsequent clinical validation, considering the aforementioned outcomes, as summarized in Figure 6. By integrating endothelium‐protective human genes, including hA20 or hHO‐1, into genetically altered donor corneas, non‐human primate xenografts are expected to demonstrate improved endothelial resistance by minimizing initial post‐transplant endothelial cell density loss by ≥ 20% at the end of 1 month, as compared to existing models. PERV‐devoid porcine‐sourced corneas are unlikely to demonstrate any PERV transfer in non‐human primate recipients 6 months post‐transplantation, as indicated by high‐efficiency metagenomic sequencing. Combined, these hypotheses constitute quantifiable and attainable routes for subsequent scientific advancement, enabling uniformity, stability, and integrative progress towards corneal xenotransplantation and ensuring therapeutic sustainability [45, 46, 47, 48]. The standard for subsequent studies is additionally contingent on clearly establishing the interaction between ethical issues and regulatory frameworks. To ensure patient safety and sustain public confidence, there must be an effective connection between legislative, administrative, and ethical obligations, specifically requiring persistent patient observation. In summary, since corneal xenotransplantation offers a potential solution to the donor shortage, additional research is required to enhance decellularization procedures and immunosuppressive approaches, and to improve corneal biomechanical adaptability and nerve regeneration capability.

Figure 6.

Associated research gaps. This figure is compiled by the authors. PERV, porcine endogenous retrovirus.

Author Contributions

Sankhajyoti Saha: conceptualization, validation, visualization, writing – review and editing, project administration, formal analysis, writing – original draft, supervision, resources, investigation, data curation. Moubani Dutta: conceptualization, funding acquisition, writing – original draft, methodology, visualization, formal analysis, project administration, software, data curation. Agnihiya Bosu: visualization, project administration, resources, writing – review and editing, methodology.

Funding

The authors have nothing to report.

Ethics Statement

The authors have nothing to report.

Consent

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

Data sharing is not applicable to this article as no new data were created or analyzed in this article.