Ocular Inflammatory Response to Intravitreal Injection of Adeno-Associated Virus Vector: Relative Contribution of Genome and Capsid

Abstract

Both subretinal dosing and intravitreal (IVT) dosing of adeno-associated virus (AAV) in higher species induce mild and transient inflammatory responses that increase with dose. Foreign protein and foreign DNA are known inducers of inflammation, which is also true in the immune-privileged ocular environment. We explored which component(s) of AAV vectors, viral capsid, or viral DNA drive inflammatory responses. Recombinant AAV with three tyrosine to phenylalanine substitutions in the capsid of AAV serotype 2 (rAAV2tYF), and with a generic ubiquitous promoter (cytomegalovirus [CMV]) controlling the expression of humanized green fluorescent protein (hGFP), was processed to enrich for AAV capsids containing genome (full capsids), capsids without genome (empty capsids), and residual material. Nonhuman primate eyes were injected by IVT in both eyes. During in-life, ocular inflammation and development of neutralizing antibodies (NAb) were measured. Following termination, lymph node immunophenotyping was performed, vitreous was processed for cytokine and RNAseq analyses, and ocular sections were assessed for transgene expression (by in situ hybridization) and histopathology. IVT dosing of AAV vectors transiently raised cellular inflammation in the aqueous and induced a more sustained inflammation in the vitreous. Lowering the total capsid dose by removing empty AAV capsids reduced inflammation and improved viral transduction. IVT dosing of AAV induced systemic NAb to AAV irrespective of the vector preparation. Similarly, lymph node immunophenotyping revealed identical profiles irrespective of viral preparation used for dosing. Immune cells in the vitreous were identified based on RNAseq analysis. Three months postdose, cytokine levels were low, indicative of minimal levels of inflammation in agreement with histopathological assessment of the retina.

Characterization of Gene Therapy Associated Uveitis Following Intravitreal Adeno-Associated Virus Injection in Mice

“…An alternative hypothesis is that inflammation is associated with specific vector variables, including the immunogenicity of specific viral capsids, high viral dose, and contamination from lipopolysaccharide, proteins, or empty viral capsids owing to different packaging protocols. 6,15,16 To address these possibilities, we used serial OCT imaging as a sensitive method for detecting anterior chamber cell and vitritis in murine eyes. 12,17,18 We also tested four different AAV preparations that varied over a range of viral doses (10 9 -10 11 viral genomes/eye), by packaging capsid (AAV2 or AAV2-7m8), cargo plasmid (see maps in Supplementary Fig.…”

“…These findings may be explained by the presence of neutralizing antibodies in the vitreous of Exposed:AAV eyes that could have impacted vector concentration and decreased early innate signaling within the eye. 15,20 Priming also influenced the timing of the adaptive immune response. Both dendritic cells and T cells were significantly more numerous in Exposed:AAV eyes than in Naive:AAV eyes on day 7.…”

Recombinant Adeno-Associated Viral Vectors (rAAV)-Vector Elements in Ocular Gene Therapy Clinical Trials and Transgene Expression and Bioactivity Assays

“…Intravitreal injections target a larger retinal area. Intravitreal injections of empty rAAV capsids can induce a temporary immune inflammation of the aqueous and the vitreous [ 303 ]. Enzymatic digestion (proteasome inhibitors) of the ILM or ILM/outer limiting membrane (OLM) or disruptions of the ILM/OLM by the disease can alter rAAV infection and allow rAAV infection deeper into the retina [ 99 , 294 , 304 , 305 ].…”

“…However, the innate, humoral, and cell-mediated immune response might contribute to vector toxicity. For example, intravitreally injected empty rAAV capsids can induce a transient inflammation of the aqueous and the vitreous body [ 303 ]. Most importantly, cis-regulatory-sequence, rAAV-capsid, and transgene-related toxicity need to be investigated in the retinal degeneration model.…”

“…Most importantly, cis-regulatory-sequence, rAAV-capsid, and transgene-related toxicity need to be investigated in the retinal degeneration model. Further, the more efficient gene expression cassettes might allow a rescue strategy at lower rAAV titers, thereby causing less toxicity and lower transient inflammation [ 303 ].…”