4 April 2023 Episode 326 Adeno-associated viruses as a cause of hepatitis and as a tool for gene therapy

Tue, 04/04/2023 - 18:18

Episode 326 Adeno-associated viruses as the cause of hepatitis in children and as gene therapy

Dear colleagues,

Remember that one year ago in April 2022, there was a sudden epidemic of severe non-A/E hepatitis in young children (usually < 10 years old).  It was not clearly linked to SARS-CoV-2 and the most likely (but non-conclusive associated was found with Adenovirus type 41f in UK (See Episode 257).  In July of 2022, a report from WHO (Ep 326-1) identified over 1000 cases, most in Europe and north-America, with only a very weak association with SARS-CoV-2 or Adeno. 46 children required liver transplantation and 22% died!


Now, there is a paper in Nature by Antonio Ho et al (Ep 326-2) showing:

  • Strong association with adeno-associated virus type 2 (AAV-2): 26/32 in cases versus 5/74 controls
  • Strong T cell infiltrate in the liver: mainly CD4+ T-cell-mediated immune pathology
  • Genetic association with HLA class II DRB1*04:01 allele was identified in 25/27 cases (93%), compared with a background frequency of 10/64 (16%; p=5.49 x 10-12).

While direct hepatoxicity of AAV has been described in gene therapy, with high doses of AAV (see below), it was not known as a cause of hepatitis in children. AAV are “depend-viruses”, which need Adeno- or Herpes virus for replication.  The burst of infection in 2022 may have been an indirect effect of the COVID-restrictions, there may have been a sudden temporary wave of primary co-infections by AAV and Adenovirus (e.g. serotype 41f) or Herpes (HHV6B, the “roseola” virus)  after the COVID restrictions were lifted.


I wanted to better understand the potential significance of this finding in the context of the popular gene therapy with AAV vectors. 

Therefore, I will first remind of the basic virology of these viruses (Par 1), provide some examples of application (Par 2); the immunology and toxicity, including the risk of inflammatory and integration damage (Par 3), with a balanced view in comparison with other formats of genetic therapy (Par 4).  

Par 1 Basic virology of AAV

Ep 326-3: John Carter Virology Textbook 2007 Chapter 12 Parvovirus

Parvoviruses are amongst the smallest known viruses, with virions in the range 18–26 nm in diameter. They

derive their name from the Latin parvus (= small).

The subfamily Parvovirinae includes the genus Dependovirus, the members of which are defective,

normally replicating only when the cell is co-infected with a helper virus.


Dependoviruses are being investigated as possible vectors to introduce genes into the cells of patients for the treatment of various genetic diseases and cancers. One of the advantages of dependoviruses

is the fact that they are not known to cause any disease  (????), in contrast to other viruses under investigation, such as retroviruses.








B) When AAV infects a human cell alone, its gene expression program is auto-repressed and latency is ensued:

-  usually exists as episome

- rarely integrates preferentially into the long arm of human chromosome 19, designated AAVS1.

A) When a latently infected cell is super-infected with a helper virus, such adenovirus or herpes x virus, the AAV gene expression program is activated leading to the AAV Rep-mediated rescue (i.e., excision) of the provirus DNA from the host cell chromosome, followed by replication and packaging of the viral genome.

Ep 326-4: Anita Meier Viruses 2020: Helper viruses of AAV

AAV are small, nonpathogenic parvoviruses, which depend on helper factors to replicate.

Provided by coinfecting helper viruses such as adenoviruses e.g. AdV5, herpesviruses e.g. HSV-1, or papillomaviruses.



Ep 326-5: Robert Shultz Mol Ther 2008: Recombinant Adeno-associated Virus Transduction and Integration


Cell entry and trafficking of recombinant adeno-associated virus (rAAV).




  • rAAV enters the cell through receptor-mediated endocytosis.
  • Trafficking to the nucleus occurs through a number of possible pathways, some of which are represented here: the endosomal processing of virions results in externalization of nuclear-localization signals and a phospholipase domain on capsid proteins, allowing endosomal escape and nuclear targeting.
  • Vector uncoating probably occurs in the nucleus, releasing the vector genome to form episomes or, rarely, to integrate into the host cell genome.
  • As judged by the transduction enhancement caused by proteasome inhibitors, proteasomes are likely to be involved in vector degradation.


Fate of recombinant adeno-associated virus (rAAV) vector genomes.



(a) Structure of an inverted terminal repeat (ITR) in the single-stranded AAV genome. Sequence elements of the ITR are labeled,

and integration hotspots are red.

(b) The single stranded rAAV genome becomes double-stranded through DNA synthesis and/or annealing. The double-stranded rAAV genome concatamerizes. The rAAV genome predominantly persists episomally. However, rare integration events do

occur, most probably at chromosomal double-strand breaks.

dsDNA,  double-stranded DNA; ssDNA, single-stranded DNA.


Authors summary:

Once inside the nucleus, the rAAV genome exists in a predominantly episomal form

→ therefore, nondividing cells tend to be most stably transduced.


However, rAAV has a low frequency of integration into the host cell genome in dividing cells often in or near genes

→ can be associated with host genome mutations


Par 2 Applications of various AAV for gene therapy


Ep 326-6: Shaza Issa Cells March 2023: Various AAV serotypes and their application



A complete list of therapies can be found in Table 2 p. 19-15


Ep 326-7: Kazuhiro Muramatsu Pediatrics and Neonatology Feb 2023: Pediatric gene therapy with AAV



Three gene therapy drugs based on rAAV vectors have been approved by the FDA and European Medicines Agency (EMA) for the treatment of diseases with limited therapeutic options:

  1. Glybera (alipogene tiparvovec) for the treatment of familial lipoprotein lipase deficiency (LPLD):

AAV1 viral vector delivers an intact copy of the human lipoprotein lipase (LPL) gene to muscle cells. The LPL gene is not inserted into the cell's chromosomes but remains as free floating DNA in the nucleus. The injection is followed by immune suppressive therapy to prevent immune reactions to the virus.


  1. LUXTURNA (voretigene neparvovec-rzyl) for patients with confirmed biallelic mutations of the RPE65 gene (i.e. patients who have inherited the mutation from both parents) and who have sufficient viable retinal cells. A recombinant AAV-2 vector with the RPE65 gene is applied subretinal. RPE65 is produced in the retinal pigment epithelial (RPE) cells and converts all-trans-retinol to 11-cis-retinol, which subsequently forms the chromophore, 11-cis-retinal, during the visual (retinoid) cycle.


  1. Zolgensma (onasemnogene abeparvovec-xioi) for the treatment of children less than 2 years with spinal muscular atrophy (SMA), associated with high mortality.  A one-time intravenous administration of Zolgensma, containg AAV-9 vector with the SMN-1 delivers the gene to the child’s motor neurons, which improves muscle movement and function, and survival. 


In addition, rAAV-based vectors are being used in clinical trials to treat many diseases, including muscular diseases (Duchenne muscular dystrophy, X-linked myotubular myopathy), hemophilia, inherited metabolic disorders (ornithine transcarbamylase deficiency, methylmalonic acidemia), neurological diseases (Parkinson’s disease,

Batten’s disease), and ocular diseases (retinitis pigmentosa, choroideremia, age-related macular degeneration)


Par 3 Immune responses and serious adverse effects

Ep 326-8: Motahareh Arjomandnejad  BioDrugs Feb 2023 Immunogenicity of AAV

Immune responses in AAV gene therapy.




AAV capsid or introduced transgene can be recognized by innate immunity which results in cytokine production, activation of innate immune cells, complement activation, and priming of adaptive immunity.

Presentation of capsid peptides or delivered transgene on MHC I molecules results in activation of CD8+ T cells and clearance of transgene expression.

Antigen presentation on MHC II activates CD4+ T cells, which leads to production of cytokines and further induces humoral or cell-mediated immune responses and creates immunological memory.

Activation of Tregs results in suppression of cellular and humoral immune responses and long-term transgene expression.


AAV adeno-associated virus, APCs antigen-presenting cells, FoxP3 forkhead box protein 3, GzmB granzyme B, IFNγ interferon gamma, MHC major histocompatibility complex, NAbs neutralizing antibodies, PFN perforin, rAAV recombinant adeno-associated virus, TCR T-cell receptor, TLR9 toll-like receptor, TNFα tumor necrosis factor alpha, Tregs regulatory T cells.







Ep 326-9: Hildegund Ertl Front Immunol 2022: Immunogenicity and toxicity of AAV.  


Various serious adverse events, especially after high dose AAV:

  • Thrombotic micro-angiopathy (TMA) with hemolytic anemia, low platelet counts, and hemolytic uremic syndrome (HUS) leading to kidney damage in 9 (out of 1400) patients treated for spinal muscular dystrophy.
  • Myocarditis in Duchenne patients
  • Fatal hepatoxicity in 4 patients with pre-existing liver disease, treated for X-linked myotubular myopathy.
  • Dorsal root ganglia toxicity (DRG) in 2 patients treated for amyotrophic lateral sclerosis


Pathogenesis: via activation of complement, cytokine storm, antibodies against the vector and/or CD8 T cells?




The graph shows the different type of immune responses that are elicited upon injection of AAV vectors.

Toxicities are underlined and listed next to the components of the immune responses that contribute or may (?) contribute to the adverse events.


Abbreviations CpG, unmethylated CpG motifs; C1 and C3, complement factors 1 or 3; DCs, dendritic cells; DRG, Dorsal root ganglia, dsRNA, double stranded RNA; MAC, membrane attack complex; MHC, major histocompatibility complex; NK cells, natural killer cells; TMA, thrombotic microangiopathy;  ? – remains to be tested in more detail.


Proposed solutions:

  • Develop more efficient vectors: higher therapeutic transgene expression
  • Avoid large single bolus with high dose AAV vectors; replace by multiple small doses
  • Perform plasmaphereses to clear circulation from AAV-antibodies
  • Provide immune suppression e.g. B cell depleting therapy



Ep 326-10: Denise Sabatino Mol Ther 2022: Consensus of  the American Society of Gene and Cell

Therapy on risk of insertional mutagenesis.  

  • Insertional mutagenesis has been reported in a small number of murine studies,
  • The risk of rAAV-mediated oncogenesis in humans is theoretical because no confirmed genotoxic events have been reported to date.


Nevertheless: While a recent case of HCC in a subject in a hemophilia B clinical trial did not find an association between the HCC and AAV integration, it raises the importance of this ongoing discussion on AAV integration and the risk of genotoxicity.


Par 4 Balancing AAV vector therapy


Ep 326-11: Pro’s and cons of AAV as vector for gene therapy


Ep 326-12: Berna Seker Yilmaz J Mother and Child 2020; Comparison with other formats of gene therapy




Non-viral e.g. liposomes






  • AAV gene therapy trials are rather popular for rare and debilitating genetic diseases without other treatment options, because it was believed that AAV themselves are not pathogenic and that the risk of integration (and mutagenesis) is low.  There is a lot of potential AAV vectors, with various potential targets and there are indeed several success stories with 3 approved therapies and many promising trials in progress.
  • However, targeting AAV to very specific cells is not evident and serious-immune based adverse events have been described.  Therefore, as shown in Ep 326-8 and -9 several strategies are being proposed to mitigate those risks, including rather heavy immune suppression. 
  • The recent finding that AAV2 can also cause serious hepatitis in young children is a warning that non-pathogenicity of the virus itself should not be taken for granted and is probably genetically determined. 
  • Alternative strategies with Adenoviruses, Retroviruses or non-viral methods have also their limitations.


Viral vectors (Adeno, AAV or lentiviruses) will have to be further modified to better target gene therapy to specific cells in order to allow dose reduction and avoid the need for immune suppression.  Personalized gene therapy will also have to take into account the genetic background and the history of potential “helper” infectious agents that could increase the risk for serious side effects.


Best wishes,