By Charlotte Hutchings
Gene therapy is an approach to treating or preventing human disorders through the delivery of therapeutic nucleic acids to diseased cells (Lundstrom, 2018). In order to achieve targeted delivery to specific organs and cell types, viruses have been employed as a gene therapy vector as they have naturally evolved to be able to enter our cells with high efficiency. Many different viruses have been explored for gene delivery, particularly adenoviruses, retroviruses (including lentiviruses), and adeno-associated viruses (AAVs), each demonstrating relative advantages and limitations. Importantly, these viruses are engineered such that they are replication deficient and unable to cause active infection in the patient (Lundstrom, 2018). However, replication deficiency alone is not sufficient to ensure the safety of these viral vectors in a therapeutic setting.
In 1999, the field of gene therapy was shaken by the first ever gene therapy death. The patient, Jesse Gelsinger, suffered from a deficiency in the ornithine transcarbamylase (OTC) enzyme and was treated with an adenoviral vector carrying a healthy copy of the OTC gene. However, Gelsinger had previously been exposed to an adenovirus meaning that his body harboured antibodies against the gene therapy vector and, therefore, mounted an extreme immunogenic reaction against the therapy. This led to multiorgan failure and death only four days after the treatment (Sibbald, 2001). Unfortunately, this is not the only example of unexpected safety problems arising from viral gene therapy vectors. The use of a gammaretroviral vector to deliver the IL2RG gene to patients suffering from X1-SCID resulted in several cases of T-cell acute lymphoblastic leukaemia. This was due to integration of the gammaretrovirus into a specific genomic site approximately 35kb upstream of LMO2, an oncogene. The presence of strong promoter/enhancer elements in the viral sequence caused upregulation of the oncogene and, consequently, cell transformation (Williams & Thrasher, 2014).
Encouragingly, AAV vectors are not believed to suffer from such safety risks because 1) AAVs are non-pathogenic and do not generate a strong immune response in humans, and 2) AAV vectors are primarily maintained as episomes rather than integrating into the host genome (Chandler et al., 2017). The latter is due to the removal of all wild-type AAV sequences from vectors, with the exception of inverted terminal repeats. This prevents site-specific integration into chromosome 19, as seen by wild-type AAV2, because this process relies on Rep proteins that are no longer encoded (Colella et al., 2018). Their excellent safety profile is one of main reasons that AAVs are currently the favoured vector for in vivo gene therapy, being used in almost 90% of gene therapy clinical trials (Cell and Gene Therapy Catapult, 2019). Indeed, two AAV-based gene therapy products are already on the market and being used to treat spinal muscular atrophy type-1 (Zolgensma), a debilitating neuromuscular disease, and Leber congenital amaurosis type-2 (Luxturna), a retinal dystrophy that causes blindness. Whilst FDA approval is taken as an indication that these therapies are safe, scientists in the field are still debating the safety of AAV vectors.
Back in 2001, a preclinical study treating mice suffering from mucopolysaccharidosis type-7 with an AAV2 vector carrying the human GUSB gene unexpectedly found hepatocellular carcinoma (HCC) in six AAV-treated mice, but none of the age-matched controls (Donsante et al., 2001). It was later found that the tumours had been caused by random integration (via non-homologous recombination) of the AAV2 vector into a genomic site called the Rian locus. As with the earlier retroviral-induced leukaemias, viral integration had resulted in dysregulation of nearby genes and subsequent cellular transformation (Donsante et al., 2007). Worryingly, a homolog of the murine Rian locus exists on human chromosome 14, and upregulation at this site has been linked to poor prognosis in HCC patients (Luk et al., 2011).
Unfortunately, the field remained confused as other large-scale studies failed to support AAV-induced tumorigenesis in mice (Bell et al., 2005; Hojun et al., 2011). Such studies, however, failed to replicate important aspects of the initial Donsante et al. experiments. Specifically, studies using adult administration of AAV, rather than neonatal delivery, were unable to detect HCC. This is likely due to the fact that the neonatal liver contains proliferating hepatocytes and has an intrinsic susceptibility to cancer. Support for this hypothesis comes from the recent finding that AAV administration to adult mice can lead to HCC in cases where the mice have liver injury or disease, a state that induces hepatocyte proliferation (Dalwadi et al., 2021). Nevertheless, the human relevance of these tumours in AAV-treated mice is still controversial.
To investigate the potential of AAV-induced tumorigenesis in humans, patients participating in an AAV2/5 gene therapy trial for acute intermitted porphyria provided liver biopsies for analysis (Gil-Farina et al., 2016). The study confirmed that AAV integration is both low in frequency and random in nature, with no clustered integration sites near genes that had been previously implicated in the mouse studies, including the Rian homolog on chromosome 14 (Gil-Farina et al., 2016). Whilst these patients will continue to be followed-up and checked for HCC, the current data is reassuring and supports the safety of AAVs as a human gene therapy vector.
That being said, in December 2020 the field received the dreaded news that a haemophilia B patient from a long-term clinical trial has developed HCC (Kaiser, 2020). The patient had been treated with an AAV5 vector carrying the Factor IX gene back in October 2019 as part of a Phase III clinical trial which was, and perhaps still is, expected to lead to FDA approval. The news comes alongside a 10-year follow-up on haemophilia A dogs treated with AAV8-FVIII (Nguyen et al., 2021). This study revealed that AAV integration had occurred in five of the treated dogs, and that this was followed by clonal expansion of integration-containing cells. Taken together these results have led to a definite unease in the field. However, the haemophilia B patient in which HCC was found suffers from chronic hepatitis B and C infection, a condition associated with over 80% of HCC cases (El-Serag, 2012). Therefore, there is still a strong possibility that the discovered tumour was not related to AAV treatment, especially given the short time period between treatment and HCC detection. Additionally, none of the dogs with clonal expansions displayed any signs of liver tumours or dysfunction (Nguyen et al., 2021).
The field is undoubtably up in the air about the safety of AAV gene therapy vectors and their potential to cause cancer. This is somewhat surprising as these viruses were previously seen to be the safest of viral vectors. Whilst there remains to be a confirmed case of AAV-induced cancer in humans, the concerning results that are beginning to emerge leaves us wondering, how did it take so long for us to realise? Are the approved AAV-based gene therapies safe for use? Do we need to take a step back and consider AAV vector design, as we did previously with adenoviral and retroviral vectors?
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