**4. Conclusion**

volumes are limited to 3–5 μL [74]. The main surgical complications observed are cataract

In humans intravitreal injections are generally performed under local anesthesia [71], by inserting a 30 gauge needle through the sclera at the pars plana region, 3.5–4 mm posterior to the limbus between vertical and horizontal muscles with limited reflux [71, 73]. In clinical

Intravitreal administration of AAV gene therapy might look tempting since it is an easier procedure with less potential surgical complications compared to the subretinal injection, especially when treating thinned degenerative retinas. However, administration of AAV intravitreally has its own caveats namely the difficulty of AAV capsids to cross the thick inner limiting membrane in the human retina and the current lack of AAV serotypes capable of transducing efficiently the human photoreceptors or RPE cells. Another obstacle is the potential AAV transduction and subsequent expression in other eye tissues, as for example, the

Pre-clinical studies in mice and rats showed that Müller glial cells can efficiently be infected after intravitreal administration of AAV2/6 or AAV2/shH10Y445F [15, 76], therefore these AAV capsids might be used to deliver CRB1 or CRB2 into Müller cells. AAV serotype shH10Y445F is however known to transduce efficiently the ciliary body epithelium when applied intravit-

The existence of 11 natural AAV serotypes and derivatives that differ in their tropism, and the different types of cells they infect, makes AAV a very useful system to infect the various cell types of the retina. The cell specificity of the AAV vector can be further increased by using cell type specific promoters, for example RPE65 or VMD2 to drive expression in retinal pigment epithelium. Or by using e.g. the rhodopsin (RH), G protein-coupled receptor kinase 1 (GRK1), 1.7-kb L-opsin promoter (PR1.7) or cone arrestin (hCAR) promoter to drive expression in rod and/or cone photoreceptors. Or using e.g. the RLBP1, GFAP or NR2E1 promoter to drive

Several pre-clinical studies showed the tropism and/or potency of the different capsids and promoter (cell specific or ubiquitous) in infecting retinal cell types such as RPE, photoreceptors and Müller glial cells. However, AAV tropism might differ *in vivo* between rodent species, dogs, non-human primates and human. AAV tropism is dependent of the route of administration, the stage of retinal development and severity of retinal dystrophy. Therefore, is quite difficult to extrapolate the data from pre-clinical studies performed in rodents directly to the human *in vivo* setting. To obtain evidence-based data for clinical gene therapy studies, researchers optimize culture protocols for human retinal organotypic cultures [77–79] or human iPSC-derived retinas to study the AAV tropism [80]. Recently, the capacity of different AAV serotypes to infect and express in human retinal cells was studied in organotypic

trials volumes between 90 and 200 μL have been injected via this route (**Table 1**).

formation due to lens-induced damage and retinal perforation [75].

128 In Vivo and Ex Vivo Gene Therapy for Inherited and Non-Inherited Disorders

ciliary body especially when using a ubiquitous promoter.

**3.3. AAV capsids and cell type specific promoters**

expression in Müller cells [17, 23, 24, 28].

really [16].

In recent years the scientific progress in the field of gene therapy for inherited retinal dystrophies culminated in the first ever approved AAV gene therapy medicine to treat LCA patients carrying mutations in the *RPE65* gene. The number of engineered AAV capsid variants and new promoters to drive expression in the different retinal cell types is raising at great speed allowing the design of more specific and more efficient viral vectors. Likewise, the number of clinical trials using AAV gene therapy is increasing at a similar rhythm, the data collected from these studies will be very useful for the development of similar therapies. Pre-clinical studies performed in mice demonstrated that AAV-mediated *CRB2* gene augmentation therapy might be a promising medicine to prevent progression of retinitis pigmentosa in patients with mutations in the *CRB1* gene. In mice at mid-stage retinal disease *CRB2* gene augmentation therapy successfully improved retinal morphology with preservation of photoreceptor cells and retinal function, therefore providing good perspectives for the forthcoming clinical trial in patients with RP due to mutations in *CRB1*.
