Gene Therapy
There is optimism that LHON may be cured, prevented, or at least reduced in severity with gene therapies. The goal of gene therapy for LHON, and other mitochondrial diseases, is to rescue mitochondrial function to an extent that is sufficient to at least relieve the symptoms of, if not cure, the target disease by supplementing intact/wild-type alleles of the dysfunctional gene above the clinical threshold for a normal physiological phenotype.
The site of the mutated mtDNA genes underlying LHON, however, is within the mitochondrial inner membrane. The mitochondrial chromosome itself is relatively simple at only 16,569 base pairs long and encoding 13 mRNAs and their associated non-coding promoter regions, as well as 2 rRNAs and 22 tRNAs for local intra-mitochondrial translation. The advantage of the genetic simplicity of mtDNA, however, is countered by its inaccessibility deep within the double-membraned mitochondrion organelle, which, normally, allows the passage of only very small molecules, such as ATP and proteins that are smaller than 10 kDa [
31]. The relative impermeability of mitochondria creates an access challenge with well-established gene delivery vectors, such as adenovirus vectors (AVVs).
To deliver a therapeutic gene product to mitochondria, one must first achieve cellular endocytosis, wherein the gene product is taken up by body cells. Secondly, one must avoid destruction by endosomes in the cytoplasm. Finally, in the most challenging problem facing researchers developing mitochondrial gene therapy approaches, one must devise a mechanism by which the gene product is made to cross into the internal space of the mitochondrion. Because nucleic acids are hydrophilic, even very small naked DNA molecules do not cross the mitochondrial membrane unaided. Given these challenges, simple physical methods that can achieve gene transfer from the extracellular space into the intracellular space, such as penetration by hydrodynamic force or bombardment, have poor efficacy for achieving targeted transfer to mitochondria within intact cells [
32].
There are a substantial variety of chemical approaches to mitochondrion-targeted gene delivery, and the approaches continue to evolve as researchers work to improve efficacy and specificity of delivery. Unfortunately, chemical approaches that rely on membrane destabilization to achieve transfer tend to be highly cytotoxic and thus not readily translatable to clinical applications [
33]. Thus, there has been substantial development of reduced-cytotoxicity cationic surfactants to carry DNA to and into mitochondria [
34,
35]. Most simply, DNA plasmids have been conjugated directly to lipophilic rhodamine carrier molecules [
36]. Efforts to improve the mitochondrion-targeting efficiency, carrier stability, and toxicity profiles of DNA carriers are ongoing. Liposome-type DNA-carrying vesicles, most notably vesicles produced from modified forms of dequalinium, have been shown to accumulate in mitochondria [
37‐
40]. There has been interest in the potential benefits of conjugating liposomes with triphenylphosphonium cation-containing molecules to improve mitochondrion targeting of carriers [
41].
There are several highly promising current lines of research aimed at developing safe and effective biological strategies for reconstituting
ND4-deficient mitochondria in RGCs with wild-type
ND4, with the long-term goal of producing a cure for LHON. Biological approaches to mtDNA delivery with ever-increasing eloquence are being developed. Some research groups have incorporated mitochondrial targeting signal (MTS) peptide tags used by cells in their strategies to deliver molecules to mitochondria, such as in MTS-conjugated peptide nucleic acid carriers [
42], MTS-conjugated lysine/histidine peptide carriers [
43,
44], and MTS-conjugated AVV carriers [
45]. This approach has been reported to provide dramatic protection from RGC loss in animal models. Notably, Yu et al. [
46] demonstrated long-term expression of
ND4, mutations of which are responsible for most cases of LHON, in mouse cells transfected with human
ND4 via an AVV carrier in which the viral capsid VP2 had been modified to include an MTS [
46].
Several research groups have worked around the mitochondrion-targeting problem by focusing on getting the
ND4 product into mitochondria after expression rather than the whole vector. This approach, known as allotopic expression, is based on the premise that
ND4 transcribed in the nucleus will be translated in the cytoplasm and then delivered to mitochondria by way of a translated MTS in the same manner that intrinsic mitochondrial proteins encoded by nuclear genes are delivered to mitochondria. Koilkonda et al. [
47] optimized a serotype-2 AVV (AVV2) carrier for intravitreal delivery of wild-type
ND4 with the MTS nucleotide sequence from the
ATP1 gene and demonstrated efficacy across a variety of experimental systems, including an in vivo mouse model, an in vivo macaque model, and ex vivo human eye model (eyes removed because of cancer). In mice, they were able to achieve allotopic mitochondrial expression of ND4 in 85% of RGCs within a week of injection. Moreover, follow-up imaging, physiological, and histological assessments demonstrated that their gene therapy strategy led to attenuation of experimental LHON model pathogenesis with respect to RGC loss, local ATP production loss, vision loss, and optic nerve atrophy [
47]. In primates, they demonstrated that their AAV2–
ND4 delivery system was well tolerated. In human eyes, they demonstrated an accumulation of allotopic ND4 protein in the mitochondria of in situ human RGCs [
48].
Employing a similar strategy, Cwerman-Thibault et al. [
49] developed an AAV2/2–
ND4 delivery system using the MTS from
COX10. They demonstrated efficient incorporation of ND4 protein into RC1 of RGCs in 8-week-old rats. Moreover, AAV2/2–
ND4-treated LHON-model rats exhibited attenuated RGC degradation and preservation of visual function [
49].
Following the aforementioned promising results, Feuer and colleagues [
50] conducted a phase I safety trial for allotopic AAV2–
ND4 gene therapy in human patients who were legally blind due to LHON caused by the 11778G>A
ND4 mutation. Of the five patients in the study, one experienced temporary minor adverse effects in the injected eye, including increased intraocular pressure and subconjunctival hemorrhage. No negative outcomes, such as further vision loss or major adverse events, were observed. Ninety days after the procedure, best corrected visual acuity (BCVA) remained unchanged in three patients, but had improved significantly in two patients [
50].
Meanwhile, researchers in the Li laboratory conducted a prospective gene therapy study in nine patients diagnosed with LHON, also due to the 11778G>A
ND4 mutation, with no spontaneous improvements in BCVA during the year preceding the study. Nine months after intravitreal injection of AAV2–
ND4 with the
COX10 MTS, significant improvements in BCVA were observed in six out of nine patients. Interestingly, patients who experienced improved BCVA in the injected eye often experienced some concurrent improvement in the contralateral non-injected eye. They conducted an accompanying experiment in mice to examine this phenomenon of contralateral improvement, the findings of which suggested it may be mediated via inter-communication at the optic chiasm [
51].
In a subsequent 36-month follow-up study of the same nine patients, no adverse outcomes were found in any of the patients. One of the patients underwent the same procedure in the other eye during follow-up, and was thus analyzed separately. Of the remaining eight patients, four experienced a significant improvement in BCVA from baseline to the 36-month follow-up time point. These four patients had a similar age of onset, LHON illness duration, and retinal nerve fiber layer thickness (both at baseline and at the 36-month time point) as the four patients who did not experience a significant improvement in BCVA, indicating that these factors could not explain patient outcomes. Temporary visual field improvements were seen in four out of eight patients, peaking between 3 and 6 months after the treatment, whereas two patients continued to show progressive visual field improvement through the 36-month time point [
52].
Moreover, GenSight Biologics, Paris, France, in three phase 3 studies on LHON demonstrated that rAAV2/2–ND4 is safe and well tolerated 2 years after a single unilateral intravitreal administration. Even so, some patients experienced early improvement on visual acuity, color vision, and contrast sensitivity in the treated eye. The most common adverse events during these studies were mild anterior chamber or vitreous inflammation and moderate intraocular pressure elevation. All ocular side effects were solved with standard therapy and no visual sequelae occurred [
53‐
55].