Chapter 23

Gene Therapy

Gene therapy entails the use of virus vectors to transfer therapeutic genes into target cells. These vectors are delivered at high concentration in solution, which are injected in close proximity to the target tissue. Currently, treatment aims primarily to correct the pathological origin of diseases with a genetic basis, and many such retinal disorders are undergoing clinical trials.

Retinal Targets for Gene Therapy

Adeno-associated virus (AAV) 2-RPE65 (LUXTURNA→) is used to treat Leber’s congenital amaurosis associated with RPE65 gene defects (LCA-RPE65); it became the first gene therapy product to obtain market approval in the U.S. and Europe. RPE65 protein is active in the retinal pigment epithelium (RPE) and is involved in the visual cycle. Replacement of RPE65 using an adeno-associated virus was shown to be safe and to improve vision in patients suffering from these conditions.

Choroideremia is an X-linked inherited retinal disease caused by mutations in the CHM gene. Current gene therapy clinical trials are assessing the safety and efficacy of subretinal AAV2-REP1, which aims to prevent disease progression by restoring the intracellular trafficking and prenylation activity functions of the Rab escort protein 1 (REP1).

X-linked retinitis pigmentosa (RP) is commonly caused by mutations in the gene encoding for the RP GTPase regulator protein (RPGR), resulting in progressive degeneration of rod and cone photoreceptors. Recent advances in AAV-mediated delivery of a functional RPGR gene have led to Investigational New Drug approvals for three AAV-based gene replacement therapies which are currently in early stages of clinical trials and are at present the most promising therapeutic approach.

Apart from monogenic diseases, common acquired diseases of the retina such as age-related macular degeneration (AMD), have become targets for gene therapy. In these types of diseases, the gene therapy is not aimed at correcting the genetic mutation responsible for the disease, but to target genes known to be involved downstream pathological pathways such as those driving neovascularisation. Gene therapy approaches for these conditions are less developed than those for inherited retinal disorders. Nevertheless, several companies are currently in phase 1 or preclinical stages of evaluating gene therapy product candidates for both wet and dry AMD. Other disease processes such as inflammation and scarring are also being targeted for possible future gene therapy.

Therapeutic Strategies of Gene Therapy

This objective is met through:

  • Restoring the missing gene, and therefore, protein function resulting from mutations in genes of the target cells – used in recessive and X-linked diseases
  • Repressing the expression of a defective gene – for dominant diseases
  • Encouraging the production of a protein to modify the original disease (e.g. neuroprotection)

Types of Available Vectors

Modified viral vectors are currently the best approach to deliver or modulate genes in target cells of various ocular tissues with very high efficiency. They can be designed to selectively infect or express in specific target cells.

The main vectors suitable for retinal cells are derived from:

  1. Adeno-associated virus - AAV vectors have the advantage of being the only vectors that can efficiently transduce both the photoreceptors and the RPE after subretinal injection. They have a relatively low immunogenicity and are tolerated in the eye. AAV vectors do not integrate into the host’s genome, persisting primarily in the nucleus of the target cells. The main limitation to AAV is its small size and therefore limited capacity in terms of gene size
  2. Lentivirus (LV) has a higher capacity but does integrate into the genome, hence carrying the risk of possible random integration into the host genome and carcinogenesis. A major drawback limiting LV use is the low transduction rate of mature photoreceptors
  3. Adenovirus (Ad) - Human Ad-based vectors were the first ones evaluated for retinal gene transfer. Ad vectors target RPE, but can cause inflammatory and immune responses resulting in damage to host cells. They are no longer widely used in trials

Surgical Delivery Method

In retinal gene therapy, virus solutions containing up to 2.0 x 1010 copies in 100μl can be injected into the required intraocular compartment. Currently, these solutions are injected into either the subretinal or intravitreal spaces. The protected immunogenicity of the eye makes gene therapy more tolerable, though immunosuppression is needed and the risk of harmful inflammatory response still exists though likely less than that which a systemic administration of the viral vector might cause. The localized approach also minimizes the required dose of vector and limits leakage into the systemic circulation, thus reducing the risk of damaging immune responses.

General Surgical Approach:

Setup – Vitrectomy Pack, 41-gauge needle/MEDONE injector system, VFC canula, intraoperative OCT (preferred but not essential).


  1. General anesthesia is preferred in young adults to avoid undesired head movements during subretinal delivery
  2. Core vitrectomy
  3. Posterior Vitreous Detachment initiation (PVD, often easier in a pediatric patient with a dystrophy than a conventional pediatric patient). Use dilute triamcinolone acetonide to confirm posterior vitreous cortex separation; consider use of pick, needle, flex loop, forceps or diamond dusted silicone tip brush as needed if the vitrectomy cutter fails to induce the PVD. Carefully detach adherent vitreous remnants from the posterior pole. (See Chapter 4 Posterior Vitreous Detachment Induction)
  4. Optional: Inject Dual Blue dye to facilitate visualization of retinotomy if re-entry through the same or an alternative site is needed. A single injection technique is recommended
  5. Peripheral vitrectomy and indented examination to rule out retinal breaks. Have a low threshold to treat suspicious peripheral lesions, lattice, tufts etc
  6. Load and prime the VFC canula with the MedOne Surgical, Inc. MicroDose&tradem; injector. Test the system using BSS. Consider bevelling the distal end of the canula at an angle of 45 degrees
  7. If available: Activate intraoperative OCT and adjust parameters to obtain a sharp scan to image the macular area (Figure 23.1A)
  8. Bleb formation:
    1. Consider bleb formation with BSS prior to the actual gene delivery subretinal injection (optional, not required)
    2. The best location for the bleb is variable. The authors recommend an initial extrafoveal retinotomy with 41-gauge needle in the supero-temporal or superior macula within the arcades (Figure 23.1B)
    3. Gently engage the needle into the retinal tissue and begin subretinal injection of the therapeutic agent by controlling the pressure with your foot pedal
    4. Keep extending the bleb until the desired area of macula is detached; this can include the fovea. More than one bleb can be created
    5. Avoid over-stretching the retina by injecting only the required amount of gene vector solution (≤300 microliters); this will avoid risk of iatrogenic macular tears. Intraoperative OCT enables safer delivery by providing direct visualization of the bleb formation and immediate changes in retinal layers
    6. The retinotomy is self-sealing and does not require retinopexy
  9. Partial fluid-air exchange
  10. Suture all sclerotomies

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