Keywords
adeno-associated virus (AAV) - biomarkers - gene therapy - thrombotic microangiopathy (TMA) - vaccine-induced immune thrombocytopenia and thrombosis (VITT)
Introduction
Gene therapy involves the administration of genetic material into a patient's body to modify specific cell functions. This methodology encompasses various approaches, such as gene replacement, silencing, editing, and addition.[1]
Replacement gene therapy delivers a functional gene to substitute for the endogenous gene carrying a loss-of-function mutation. Considerable progress has been achieved in recent years in replacement gene therapy with encouraging results for the treatment of monogenic life-threatening or highly incapacitating disorders. So far, replacement gene therapies that have been granted marketing authorization by the European Medicine Agency (EMA) and the Food and Drug Administration (FDA) include alipogene tiparvovec for patients with lipoprotein lipase deficiency (withdrawn by EMA in 2017 due to lack of demand), voretigene neparvovec-rzyl for Leber's congenital amaurosis, onasemnogene abeparvovec for spinal muscular atrophy (SMA), fidanacogene elaparvovec (approved by FDA) and etranacogene dezaparvovec (conditionally approved by the EMA) for hemophilia B, and valoctocogene roxaparvovec for hemophilia A (conditionally approved by the EMA).[2] All these gene therapies rely on recombinant adeno-associated virus (AAV) as vector for gene delivery highlighting the versatility and usefulness of this viral vector platform.[1]
[3]
[4] Moreover, several clinical trials evaluating AAV-based gene therapies are currently ongoing. A search of the database clinicaltrials.gov performed on April 20, 2024 with the keyword “AAV” identified 116 active (recruiting or nonrecruiting) studies, including trials on diseases such as Parkinson's disease, age-related macular degeneration, and Fabry's disease.
AAVs are nonenveloped viruses that rely on other viruses for their replication and, although they can infect humans, have not been linked to any human diseases.[1] Several favorable characteristics of AAVs have led to the development of AAV-based vectors for gene therapy, like mild immunogenicity in humans, wide-ranging tropism, virtually no integration in the host genome, prolonged permanence in the host as nuclear extrachromosomal episomes, and long-lasting gene expression.[1]
[5]
[6] Despite these advantages over other viral vectors, and the successful development of several gene therapies, AAV-based gene delivery is not without limitations. In fact, beside the considerable improvements obtained with gene therapies with an overall positive impact on patients' clinical outcomes and quality of life,[7] safety issues must be carefully appraised.[8]
[9] Immune responses against the vector remain a major challenge as they can reduce treatment efficacy and cause adverse reactions.[5]
[10]
[11] In particular, adaptive immune responses against the virus capsid and the transgene product can impair replacement gene-therapy by reducing the effectiveness of cell transduction with the AAV-vector and the amount of available transgene product. At the same time, innate immunity involving the complement system is increasingly implicated in some severe adverse events, including thrombotic microangiopathy (TMA) reported in patients treated with gene therapy.[5]
[10]
[11] In clinical trials of AAV gene therapy, severe treatment-related adverse events have been reported with a rate of 30.6%, according to a meta-analysis of 255 studies.[12] These events included hepatotoxicity, dorsal root ganglia toxicity, myocarditis, and TMA.[6]
[10]
[12] In particular, hepatotoxicity and TMA are consistently associated with systemic AVV gene therapies[6]
[12] and appear to be dependent on AAV-vector doses and to the route of administration (i.e., tissue-specific responses at the site of injection, systemic immune responses in case of intravenous injection).[10] Predictors of severe adverse events have yet to be established; however, possible candidates include gene therapy characteristics, such as vector dose, and patient characteristics, such as pre-existing immunity to AAV, disease stage, and body weight.[6]
TMA is an overarching term used to describe a disease process as well as a heterogeneous group of rare disorders characterized by vessel occlusion, thrombocytopenia, and microangiopathic hemolytic anemia,[13]
[14]
[15]
[16] the latter being the hallmark of this condition. In particular, microangiopathic hemolytic anemia is characterized by many schistocytes and red blood cell fragments, high lactate dehydrogenase levels, and low/absent haptoglobin. TMA, regardless of its etiology, is a hematologic emergency that requires urgent treatment, being associated with considerable morbidity and mortality.[17]
[18]
[19] The classification of TMA is complex and continuously evolving and, so far, no shared and consistent terminology is available for defining the various members of the TMA family.[20] Drug-related TMA may be included in the larger group of thrombosis with thrombocytopenia syndrome (TTS), which encompasses a heterogenous group of immune and nonimmune conditions including cancer, thrombotic thrombocytopenic purpura (TTP), disseminated intravascular coagulation (DIC), and antiphospholipid antibody syndrome.[21] TTS diagnosis is not based on specific test results and is readily applicable also to low-resource settings,[22] thus it is especially used by the World Health Organization and regulatory agencies in the context of pharmacovigilance. Recently, a new disease called vaccine-induced immune thrombocytopenia and thrombosis (VITT) has been identified and included among the immune-mediated TTS.[23]
[24] This novel syndrome emerged in March 2021 at the beginning of the vaccination campaign against coronavirus disease 2019 (COVID-19), and was reassociated with the administration of the first dose of the adenovirus (AV)-based vaccines ChAdOx1 nCoV-19 and Ad26.COV2.S against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).[25]
[26]
[27]
This article aims to review current literature about TMA-related adverse events associated with AAV-based gene therapy, with the ultimate goal of discussing strategies to optimize the safety of this innovative and promising therapeutic approach. The review will first focus on TMA characteristics and on reports of TMA associated with AAV-based gene therapy. Then it will discuss the proposed mechanisms responsible for these events, comparing them with those leading to VITT, given some similarities between these two syndromes, to properly position them in the heterogenous group of drug-related TTS. However, despite some “similarities,” VITT is clearly not a TMA disorder. Heparin-induced thrombocytopenia (HIT) and VITT typically cause large-vessel thromboses involving veins and arteries, whereas organ dysfunction in TMA typically involves microvascular platelet-rich microthrombi. The existence and influence of cognitive bias in medicine is a well-known topic and is gaining increased attention.[28]
[29] Given that “recency bias” may lead to choosing a particular interpretation or diagnosis because it is at the front of mind,[29] it is worth considering that AAV gene therapy–related thrombotic disorders may be confused for VITT by nonexperts in coagulation disorders, considering that the latter has dominated the drug safety literature in the last few years. Accordingly, a clarification of the specific characteristics and mechanisms of the two distinct adverse events related to these innovative but different virus-based platforms, namely AAV for gene therapy and AV for the COVID-19 vaccine, may be useful to provide the knowledge and rationale for appropriate clinical management. [Table 1] provides a summary of the main TTS discussed in the present review, along with their clinical characteristics and diagnostic criteria, classifying them as being a TMA or not. Finally, potential biomarkers for TMA diagnosis and for the identification of patients at risk of TMA will also be addressed.
Table 1
Comparative characteristics of some TTS
|
Disease
|
TMA
|
Causes
|
Clinical presentation
|
Laboratory diagnostic criteria
|
|
|
TTP
|
Yes
|
Failure to degrade ultra-large multimers of von Willebrand factor
|
● Severe thrombocytopenia (platelet count < 30 × 109/L)
● Microangiopathic hemolytic anemia
● New focal neurologic symptoms, seizures, or myocardial infarction (multisystem involvement)
● Kidney injury mild or absent
|
ADAMTS-13 deficiency (<10% of normal or <10 IU/dL)
|
|
|
HUS
|
Yes
|
● Exposure to Shiga toxin (classic HUS)
● Defect in complement factors (complement-mediated HUS or atypical HUS)
|
● Thrombocytopenia
● Microangiopathic hemolytic anemia
● Kidney injury (predominant feature)
|
● Shiga toxin in stool (classic HUS)
● Genetic testing (complement-mediated HUS)
|
|
|
TMA associated with AAV-based gene therapy
|
Yes
|
Exposure to AAV-vector
|
● Thrombocytopenia
● Microangiopathic hemolytic anemia
● Kidney injury
● Myocardial inflammation
● Hepatic toxicity
|
ND
|
|
|
HIT
|
No
|
Heparin treatment
|
● Thrombocytopenia of moderate severity with onset at 5–14 days after heparin initiation
● Thrombosis
|
● Normal platelet count before heparin initiation
● Thrombocytopenia defined as a drop in platelet count by 30% to <100 × 109/L or a drop by >50% from the patient's baseline platelet count
● Positive test for HIT antibodies (including platelet activation test)
|
|
|
VITT
|
No
|
Exposure to AV-based anti-SARS-CoV-2 vaccines (leading to anti-PF4 antibody production)
|
5–30 days after vaccination:
● New severe headache, not responding to simple analgesia
● Unusual headache that is worse when lying down, or associated with new blurred vision, speech difficulty, motor weakness, drowsiness, or seizures
● New unexplained pinprick bruising or bleeding
● Shortness of breath, chest pain, leg swelling, persistent new abdominal pain
|
● Thrombocytopenia (platelet count < 150 × 109/L)
● D-dimer level > 4,000 μg/L FEU
● Positive test for anti-PF4 antibodies (including platelet activation test)
|
|
Abbreviations: ADAMTS-13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; EIA, enzyme immunoassay; FEU, fibrinogen equivalent unit; HIT, heparin-induced thrombocytopenia; HUS, hemolytic uremic syndrome; ND, not defined; PF4, platelet factor 4; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TM, thrombotic microangiopathy; TTP, thrombotic thrombocytopenic purpura; VITT, vaccine-induced immune thrombotic thrombocytopenia.
Note: References[14]
[15]
[22]
[27]
[31]
[32]
[44]
[51].
Classification of Thrombotic Microangiopathies
Classification of Thrombotic Microangiopathies
An established classification of TMA disorders is lacking, according to the complexity of the scenario and its rapid evolution. Clinically, TMA disorders are characterized by thrombocytopenia, microangiopathic hemolytic anemia, and brain and kidney injury, although injury to any other organ may also occur[14]
[15] (Visual summary). Thrombocytopenia arises from the consumption of platelets into microcirculatory platelet-rich thrombi, while microangiopathic hemolytic anemia is caused by the fragmentation of red blood cells in the occluded microvasculature.[15] Kidney injury is frequent, as this organ is particularly vulnerable to microvascular occlusion and endothelial dysfunction.[13] Two members of the TMA group with these characteristics—TTP and the hemolytic-uremic syndrome (HUS)—have been known for decades.[14] The pathogenic mechanisms underlying TTP and HUS were discovered in the early 1980s, namely the inability to degrade ultra-large multimers of von Willebrand factor (VWF) for TTP, and the exposure to the Shiga toxin produced by Escherichia coli leading to endothelial damage for HUS.[30]
[31] Acquired and congenital TTPs are due primarily to severe ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) deficiency; atypical HUS (aHUS) is commonly associated with complement dysregulation and Shiga toxin, drugs, immune complexes, and others agents likely damaging the endothelium. Impaired post-secretion processing of VWF due to deficiency of ADAMTS-13 (immunoglobulin G [IgG] antibodies or congenital), dysregulation of the alternative complement pathway (mutations and/or specific antibodies), and endothelial injury are pathophysiologic mechanisms involved in these TMAs.[14]
TMA disorders are currently divided into primary and secondary forms. Primary TMAs are associated with genetic or acquired factors related to specific mechanisms (i.e., low or undetectable ADAMTS-13 activity for TTP, permanent complement activation for aHUS); while in secondary forms, other diseases or drugs, by different mechanisms, cause the activation of complement,[10]
[32] finally leading to microvascular endothelial injury (e.g., AAV-associated TMA).
Drug-induced TMA has been reported in association with several medicinal products.[33] A systematic review of 344 reports involving a total of 586 patients identified 78 drugs implicated in drug-induced TMA.[34] Of these, 22 (28%) were conclusively demonstrated as causing TMA, while 20 (26%) were likely associated with TMA.[35] Clinical presentation of drug-induced TMA is variable, including organ damage, neurological signs, and ischemia, suggesting different pathogenic mechanisms which can involve a direct toxic effect or an immune-mediated effect.[13] In immune-mediated drug-induced TMA, disease onset is usually acute and drug-dependent antibody testing may help identify the causative agent. In nonimmune drug-induced TMA, the onset is usually gradual and dose-dependent.[20]
In a review article on the mechanisms of TMA published some years ago, three types of microangiopathy were recognized based on histopathologic features: TMA with systemic platelet thrombi (for example TTP), TMA with predominantly renal platelet-fibrin thrombi (for example classic and familial HUS), and TMA with renal or systemic thrombi (for example, drug-induced TMA).[14]
TMA Associated with AAV-Based Gene Therapy
TMA Associated with AAV-Based Gene Therapy
We searched PubMed for “TMA AND [adeno-associated virus OR AAV]” and “thrombosis AND [adeno-associated virus OR AAV]” and “[TMA OR thrombosis] AND gene therapy,” selecting case reports or case report reviews describing the adverse event of interest up to March 2024.
There is still very limited information concerning TMA in patients receiving AAV-based gene therapy. This is primarily due to the small number of patients enrolled in clinical trials evaluating gene therapy for the treatment of rare diseases together with the very low incidence of this adverse event. With respect to the reporting of adverse events related to gene therapy (e.g., for the treatment of Duchenne muscular dystrophy [DMD]), substantial efforts toward prompt disclosure have been made by pharmaceutical companies producing AAV-based gene therapies in compliance with the request of regulatory authorities and safety issues have been addressed, whenever possible, in a multidisciplinary approach, with the engagement of the scientific community.[36]
TMA has been initially reported with gene therapy products that use the vector serotype AAV9 delivered systemically and at high doses (≥5 × 1013 vector genomes/kg body weight), for the treatment of neuromuscular disorders in children[6]: onasemnogene abeparvovec approved for the treatment of SMA, and SGT-001 and PF-06939926 under investigation for the treatment of DMD[6]
[33] [NCT03368742, NCT03362502]. For two patients (one treated with onasemnogene abeparvovec and the other with SGT-001), TMA was fatal.[10]
[20]
[37]
[38] So far, no TMA-related events have been associated with voretigene neparvovec-rzyl for the treatment of inherited retinal dystrophy, which uses an AAV2 vector and is delivered via a subretinal injection.[6]
TMA in children is a rare event (∼1.0–3.3 cases/million/year).[33] According to an FDA report published in 2021 and addressing AAV-related TMA, nine cases of TMA were reported over more than 1,400 patients treated with onasemnogene abeparvovec; these cases involved children aged between 4 months and 4 years.[33] In four published case reports, TMA symptoms started shortly after the beginning of treatment (at day 8) and the patients had hematologic markers of complement activation (low C3 and C4 and/or increased levels of soluble C5b-9 complex).[33]
[37]
[39] More recently, complement activation has been reported in adult subjects with Fabry's disease receiving one single dose of 4D-310 (AAV2 capsid variant) in combination with prophylactic oral corticosteroids, thus suggesting that these phenomena are likely not AAV9-specific.[40] In the majority of cases, TMA eventually resolved after management including fluid and electrolyte infusion, platelet or red blood cell transfusion, dialysis, plasmapheresis, and eculizumab,[33] while one case was fatal ([Supplementary Table S1], available in the online version).[37]
Importantly, to optimize the gene therapy safety profile, several preventive/therapeutic strategies targeting the immunologic response to AAV have been proposed, including (1) high-dose glucocorticoids; (2) anti-CD20 mAbs that, by the depletion of B cell populations, impair antibody production over time; (3) mTOR inhibitors that contribute to the inhibition of T and B cell activation; (4) plasmapheresis, and (5) cleavage of circulating IgG antibodies.[41]
[42]
Possible Mechanisms of TMA Associated with AAV-Based Gene Therapy
Possible Mechanisms of TMA Associated with AAV-Based Gene Therapy
The mechanisms leading to the TMA-related events reported with AAV-based gene therapy are not fully elucidated. Recent research work has focused on the role of the innate immune system in the response to AAV-based gene therapy.[43] The involvement of the innate immune system has been suggested by the consistent evidence of complement-mediated events in the few reports of TMA discussed above.[20]
[33]
[37] Complement may be activated by the classical (Ig-mediated) and alternative pathways, which converge to produce the C3 convertases that cleave C3 into C3a and C3b, the latter binding C4b2b to create C5 convertase. The C5 convertase produces C5a, a very potent inflammation mediator, and C5b, which in turn binds to C6, C7, C8, and C9 to generate the C5b-9 membrane attack complex mediating cell death.[44] Complement pathways, which are components of the innate immune system, protect against viruses and can interact with the adaptive immune system. Activation of these pathways initiates a cascade of events—inflammatory response, recruitment of neutrophils, stimulation of coagulation, endothelial cell damage, and platelet activation—that eventually result in injury to the microvasculature and thrombosis.[10] Innate immune responses are rapid (within a few days to 2 weeks from exposure to the virus, as observed in the few reports of TMA in patients treated with AAV gene therapy), while antigen-specific adaptive immune responses develop usually after several weeks.
A few studies have attempted to dissect the pathogenesis of TMA related to AAV gene therapy.[43]
[45] Detailed studies on TMA mechanisms are still lacking, also because in animal models a direct involvement of the complement system in AAV-mediated events has not clearly emerged.[4] Importantly, it has been demonstrated that AAV capsid particles interact with some complement proteins (namely C3, C3b, iC3b) and complement regulatory factor H.[41] More recently, to bypass possible species differences, Smith et al have performed studies using human whole blood and have characterized the innate immune response to AAV.[43] They found that AAV particles were mainly internalized by neutrophils, monocyte-related dendritic cells, and monocytes. Low titers (≤1:10) of pre-existing AAV-neutralizing antibodies had a negligible effect on the innate immune response to AAV, while higher titers (≥1:100) were associated with a significant increase in the secretion of proinflammatory cytokines, AAV-vector uptake by antigen presenting cells, and complement activation. The results of this study suggest that pre-existing antibodies to AAV vectors may play a crucial role in the immunogenicity of AAV-based gene therapies and that inhibition of the complement pathway may be a strategy for improving the safety of these therapeutics.[43] West et al investigated complement activation by AAV9 in sera from seronegative and seropositive (i.e., carrying neutralizing antibodies from a previous environmental exposure to AAV) human donors.[45] The interaction of AAV9 with seropositive sera resulted in complement activation; again, complement activation was associated with the sera that had higher levels of anti-AAV IgG1 antibodies. These findings suggest that immunoglobulin depletion might be a strategy for reducing the risk of complement activation associated with AAV-based gene therapy.[45] More importantly, clinical data have been recently published in support of the involvement of complement activation, corroborating its possible targeting for therapeutic purposes. Salabarria et al reported that, after the systemic AAV9 administration to deliver gene therapies, subjects treated with only corticosteroids (n = 13) showed an increase of IgM, IgG, D-dimer, a decline in platelet count, and direct and alternative complement activation (depletion of C4, elevated SC5b-9, Ba, and Bb antigens). On the other hand, subjects treated with corticosteroids plus rituximab and sirolimus (n = 25) did not show significant changes in either the Ig profile or in complement levels.[40] Hence, despite some limitations, such as the absence of randomization and the heterogeneity of clinical diagnoses, the study has shown that TMA in the setting of AAV-gene therapy is primarily due to complement activation by the classical (antibody-dependent) pathway, possibly amplified by the alternative pathway.
Does AAV Gene Therapy–Related TMA Share Mechanisms with VITT and Other Immune-Mediated TTS?
Does AAV Gene Therapy–Related TMA Share Mechanisms with VITT and Other Immune-Mediated TTS?
The emergence of TMA associated with AV-based anti-SARS-CoV-2 vaccines has prompted intensive research on the pathogenic mechanisms of these thrombotic events.[23]
[46]
[47]
[48]
[49] Currently, it does not seem possible to completely exclude that VITT and AAV-vector–related TMA may share some features, and the efforts toward the understanding of VITT may contribute to improve current knowledge of the mechanisms leading to severe events in patients treated with AAV-based gene therapy, at least as a reference model from which the latter differentiate.
The evolution of VITT denomination is a clear example of how complex is the classification of thrombotic disorders, even in the current era where disease nomenclature has progressively increased its role in medicine.[49] Indeed, while the first paper reporting the syndrome used “thrombotic thrombocytopenia” in the title, this name was later considered misleading as VITT is not a TMA, i.e., schistocytes are absent and, if present, in keeping with an underlying DIC. Accordingly, most of the subsequent papers adopted the same acronym but as “vaccine-induced immune thrombocytopenia and thrombosis,” paralleling “heparin-induced thrombocytopenia and thrombosis” (HIT/T),[50] which also is not a TMA. In addition, HIT and VITT typically cause large-vessel thromboses involving veins and arteries, whereas organ dysfunction in TMA disorders is typically the consequence of microvascular-platelet-rich microthrombi.
Although considerable progress has been made in the understanding of VITT, the suggested pathogenesis remains, at least in part, unclarified.[20]
[27]
[49]
[51]
[52] VITT has been classified in the larger group of “platelet activating anti-platelet factor 4 (PF4) disorders” including diseases characterized by (1) thrombocytopenia and thrombosis; (2) circulating anti-PF4 antibodies; (3) a short time interval from the initial exposure to the trigger (heparin; AV) to clinical manifestations; and (4) clotting activation mediated by the activation not only of platelets, but also of monocytes, neutrophils, and endothelium.[53] Patients with VITT present antibodies directed to PF4, which are pathogenic and induce platelet activation and the formation of neutrophil extracellular traps leading to thrombosis and thrombocytopenia.[48] The formation of anti-PF4 antibodies is thought to be caused by the presence of PF4-vaccine complexes which, along with the proinflammatory environment caused by vaccination, can trigger a B cell response.[48] According to this view, VITT is a condition similar to autoimmune HIT, a potentially devastating reaction that can occur also without previous exposure to heparin and is caused by antibodies against PF4,[27]
[48] although the target antigen(s) on PF4 are different between HIT and VITT antibodies.[53]
It has been recently suggested to use the term “autoimmune HIT” in situations where heparin is the initiating trigger, but in which the resulting antibodies can strongly activate platelets even in the absence of heparin while HIT-like disorders triggered in the absence of heparin should be called “spontaneous HIT.”[54] Importantly, both HIT-like and VITT-like antibodies can be triggered in the absence of proximate heparin or vaccine exposure.[55]
[56] It is worth mentioning that AV infections had already been reported to be associated with thrombotic disorders,[57] but that recently it has become apparent that they can trigger a VITT-like syndrome characterized by anti-PF4 antibodies with platelet-activating properties, severe thrombocytopenia, extreme D-dimer elevation, hypofibrinogenemia, and multiple atypical thromboses, including cerebral venous sinus thrombosis.[56]
[58]
[59]
[60] Thus, PF4/polyanion ELISAs are considered a good screening test for both HIT/HIT-like and VITT/VITT-like antibodies.[61]
PF4 is a soluble protein that can form dimers and tetramers. Positively charged PF4-mers avidly bind to negatively charged heparin (a polymeric anion). PF4–heparin complexes undergo conformational changes and can become ultra-large and highly immunogenic.[48] In HIT, antibodies to PF4–heparin complexes can activate platelets via the binding to FcγRIIA receptors, leading to the formation of microthrombi and large-vessel thrombosis.[49]
[51]
[62] It is hypothesized that the VITT counterparts of the highly immunogenic PF4–heparin complexes seen in HIT are complexes of PF4 with DNA and polyadenylated hexon proteins of the AV vector that could come in contact with blood during vaccine administration.[20]
[49]
[62] Both vector components could provide a scaffold of negative charges similar to heparin.
In conclusion, AV vectors can induce an immunologic reaction that results in the production of high-titer anti-PF4 antibodies; these, in turn, cause the thrombotic events associated with VITT. An interesting question that deserves further investigation is which additional anionic molecules may be involved in the formation of complexes with PF4, besides those already identified.[52]
[63]
[64] However, until today no evidence has been collected about the possible involvement of these mechanisms (i.e., anti-PF4 antibodies with strong platelet-activating properties, properly assessed by experienced reference laboratories) in the TMA related to AAV gene therapy, in agreement with the different mechanism of action, with the obvious consequences for possible mechanism-driven therapeutic options.
Biomarkers
Currently no biomarkers to identify patients at risk have been validated for either TMA or VITT. It should however be pointed out that while biomarkers related to an increased risk for a severe adverse event would be crucial for small populations of patients candidate to therapy, like those potentially treatable by gene-therapy approaches, they would not be cost-effective to screen thousands of healthy subjects candidate to vaccination. On the other hand, biomarkers useful for TTS diagnosis and monitoring would have similar relevance in the two clinical settings. Several useful laboratory parameters have been identified. For example, plasma ADAMTS-13 activity measurements have been shown to be essential not only for the initial diagnosis of immune TTP, but also for risk stratification and prompt detection of disease progression or relapse.[65] Of note, thrombocytopenia and platelet activation are characteristic of many different TTS conditions, highlighting the need for disease-specific tools for the assessment and monitoring of changes in platelet activation and function in different settings. Increased soluble C5b9 has been reported in complement-related diseases, including C3 glomerulonephritis and aHUS, and has been proposed as a biomarker of disease progression in HUS and TMA associated with hematopoietic stem cell transplantation.[37]
In the field of gene therapy, there is an urgent and widely recognized need for biomarkers to identify patients at high risk of TMA complications before initiating AAV-based gene therapy, and to ensure the prompt recognition and diagnosis of TMA following therapy delivery.[36]
[40]
[53]
[62]
[66] Predisposing factors for TMA to be considered before initiating gene therapy include infections, anti-factor H antibodies, defects in ADAMTS-13 activity, genetic susceptibility to HUS, and increased levels of sC5b9 (a biomarker of complement activation).[36] Patients with biallelic null mutations in the gene to be replaced and no cross-reactive immunologic material (i.e., CRIM-negative) are naïve to the transgene product and have a high risk of developing an immune response to it due to the lack of immune tolerance; this could lead to adverse events and ineffective therapy.[37] Notably, none of these factors have so far been validated for the selection of candidates to gene therapy. Importantly, the total anti-AAV Ab levels should be measured before and after AAV gene therapy administration, because assessing only the neutralizing antibodies provides scarce information about the total amount of complement-activating antibodies (e.g., IgM).[67]
In the posttreatment setting, current safety indications about gene therapy issued by the EMA recommend stringent monitoring of platelet count in the weeks following therapy delivery.[66] Screening for hemolysis after any decrease in platelet counts is also recommended. Troponin increases, uremia, increased D-dimers (suggesting blood clotting activation), anemia and schistocytes on the blood smear should also be assessed.[6] In case of clinical suspicion, anti-PF4 antibody assays (PF4/polyanion ELISAs) should be performed to rule out HIT/VITT.[61] The recent clinical data from Salabarria and colleagues reaffirm the relevance of frequent immunosurveillance during the first 30 days post-AAV administration, including not only D-dimer, but also comprehensive complement and hematology panels, in addition to other biomarkers of endothelial activation.[40]
Discussion
Replacement gene therapy is a valuable approach to the treatment of rare, life-threatening conditions for which no other therapeutic options are available. In recent years the progress with AAV-based gene therapy has been considerable leading to the approval of several gene therapies. As with any other treatment, replacement gene therapy is associated with possible adverse events, and the assessment of individual risk–benefit balance should be considered. The reported adverse events, and in particular TMA, appear to be due to the complex set of immune responses elicited by the AAV-vector.
Evidence concerning AAV-related TMA is currently very limited, although increasingly emerging. There is therefore an urgent need to improve and further encourage case reporting and data sharing among clinicians.
Elucidating the mechanisms leading to AAV-related adverse events is crucial for implementing preventive measures, identifying patients at risk (biomarkers needed), adjusting therapy (dosage, improving vectors toward a more effective and safer gene delivery), and managing the adverse event when not preventable. The identification of the complement system as a crucial player in TMA has important therapeutic implications for patients receiving AAV-based gene therapy due to the availability of already validated complement inhibitors and other drugs able to indirectly prevent its activation.[40]
Although TMA is a rare condition, it can occur in many settings and its consequences can be devastating. There is a need to increase the awareness of these disorders. To this purpose, nomenclature and classification should be consolidated and standardized. Consistent safety monitoring guidelines are required as well.
Conclusion
AAV gene therapy is fundamental for people, very often children, affected by rare, fatal or highly incapacitating conditions. The potential benefits of this therapeutic strategy are relevant. Additional efforts directed to the further improvement of AAV-based replacement gene therapy are needed.
To enhance the safety and effectiveness of AAV gene therapies, it is crucial to implement a comprehensive approach addressing the complexities associated with treatment-induced TMA. First and foremost, a concerted effort to improve awareness and education about TMA among health care providers and researchers is needed. A pivotal step in this direction involves establishing a clear and universally accepted nomenclature and classification system for TMA, including disorders associated with AAV gene therapy. Concurrently, the development of shared guidelines for the diagnosis and management of AAV-induced TMA is imperative. These guidelines should encompass safety monitoring protocols and emphasize the implementation of immune-monitoring for patients undergoing AAV gene therapy.[11] Additionally, further research should be directed toward understanding the intricate mechanisms behind AAV-induced TMA. This involves exploring the underlying biological processes that lead to TMA and searching for relevant biomarkers that can aid in diagnosis and risk assessment. Moreover, it is essential to focus on the development of innovative strategies aimed at addressing the diverse issues related to AAV immunity.[5]
[68] Importantly, recent evidence reinforces initial data about the existence of already available preventive/treatment options that may be safely used to prevent TMA.[40]
[43] By collectively pursuing these initiatives, the scientific community can optimize the administration and follow-up of AAV gene therapies, ultimately benefiting patients worldwide.
