Safety and effects of the vector for the Leber hereditary optic neuropathy gene therapy clinical trial

Abstract

IMPORTANCE We developed a novel strategy for treatment of Leber hereditary optic neuropathy (LHON) caused by a mutation in the nicotinamide adenine dinucleotide dehydrogenase subunit IV (ND4) mitochondrial gene. OBJECTIVE To demonstrate the safety and effects of the gene therapy vector to be used in a proposed gene therapy clinical trial. DESIGN AND SETTING In a series of laboratory experiments, we modified the mitochondrial ND4 subunit of complex I in the nuclear genetic code for import into mitochondria. The protein was targeted into the organelle by agency of a targeting sequence (allotopic expression). The gene was packaged into adeno-associated viral vectors and then vitreally injected into rodent, nonhuman primate, and ex vivo human eyes that underwent testing for expression and integration by immunohistochemical analysis and blue native polyacrylamide gel electrophoresis. During serial follow-up, the animal eyes underwent fundus photography, optical coherence tomography, and multifocal or pattern electroretinography. We tested for rescue of visual loss in rodent eyes also injected with a mutant G11778A ND4 homologue responsible for most cases of LHON. EXPOSURE Ocular infection with recombinant adeno-associated viral vectors containing a wild-type allotopic human ND4 gene. MAIN OUTCOMES AND MEASURES Expression of human ND4 and rescue of optic neuropathy induced by mutant human ND4. RESULTS We found human ND4 expressed in almost all mouse retinal ganglion cells by 1 week after injection and ND4 integrated into the mouse complex I. In rodent eyes also injected with a mutant allotopic ND4, wild-type allotopic ND4 prevented defective adenosine triphosphate synthesis, suppressed visual loss, reduced apoptosis of retinal ganglion cells, and prevented demise of axons in the optic nerve. Injection of ND4 in the ex vivo human eye resulted in expression in most retinal ganglion cells. Primates undergoing vitreal injection with the ND4 test article and followed up for 3 months had no serious adverse reactions. CONCLUSIONS AND RELEVANCE Expression of our allotopic ND4 vector in the ex vivo human eye, safety of the test article, rescue of the LHON mouse model, and the severe irreversible loss of visual function in LHON support clinical testing with mutated G11778A mitochondrial DNA in our patients.

Figure 1. Expression of Wild-Type (WT) Human ND4

Confocal microscopy of a retinal flat mount focused on the retinal ganglion cell (RGC) layer of the ex vivo human eye infected with WT ND4FLAG delivered by a triple Y-F capsid–modified self-complementary adeno-associated viral (scAAV) vector shows cell nuclei labeled by 4′,6-diamidino-2-phenylindole (DAPI) (A) and the anti-FLAG antibody (B) around the nuclei (C). At higher magnification, many DAPI nuclei are seen (D), with 1 cell filled with FLAG (E) surrounding the nucleus (F). High magnification of a FLAG-positive cell in the RGC layer (G) reacted with the mitochondrial membrane protein antibody against porin (H) reveals colocalization of ND4FLAG with the mitochondrial marker (I). A bar plot (J) shows counts of ND4FLAG-labeled RGCs in the ex vivo human eye. Vg indicates vector genome.

Figure 2. Transcription and In Vivo Rescue Effects of Human ND4

Reverse transcription polymerase chain reaction (A) of RNA isolated from the retinas and optic nerves (ONs) of 10 mice that received injections of mutant and wild-type (WT) allotopic ND4 into the right and left eyes showed the expected 242–base pair (bp) bands. The ND4 plasmid served as a positive control. Heart tissue isolated from injected animals was negative for human ND4. Gene sequencing of the amplicons (B and C) showed R340H mutant (MT) allotopic ND4 (CAC-histidine) and R340RWT allotopic ND4 (AGG-arginine) DNA to be present. ATP indicates adenosine triphosphate. For rescue of visual dysfunction, a bar plot (D) shows the percentage of worsening pattern electroretinogram (PERG) amplitudes of mock-treated left eyes relative to those of normal (no injection) controls was statistically significant, but no differences were detected between the treated right eyes and controls. Results of in vivo imaging (E-L) indicate that eyes protected against the effects of R340H MT ND4 by WT allotopic ND4 packaged in triple Y-F capsid–modified self-complementary adeno-associated viral (scAAV) vector showed no evidence of optic nerve head swelling or loss of the inner retinal layers at 1 (E), 3 (F), 6 (G), and 12 (H) months after injection. In contrast, the mock-treated left eyes developed swelling of the optic nerve head (arrowheads) (I), with progressive thinning of the inner retina at 3 (arrowheads) (J) and 6 months (arrowheads) (K) after injection and almost complete loss 12 months after injection (L). GCL indicates ganglion cell layer; INL, inner nuclear layer; and ONL, outer nuclear layer. A bar plot (M) shows that differences in thickness of the retinal ganglion cell and inner plexiform layers (RGC + IPL) between the rescued right eyes and mock-treated left eyes were statistically significant by 6 and 12 months after injection. Whiskers indicate standard error. ^aP = .02. ^bP = .006. ^cP = .005

Figure 3. Histopathologic Findings of the Retina

One year after intravitreal injections, low- (A), medium- (B), and high- (C) magnification hematoxylin-eosin–stained transverse sections of mice eyes that received double injections with self-complementary adeno-associated viral (scAAV2) vector wild-type (WT) allotopic ND4FLAG and single-stranded AAV2 (ssAAV2) mutant allotopic R340H ND4FLAG had an intact retinal ganglion cell (RGC) layer (arrowheads). In contrast, the mock-treated left eyes showed loss of the inner retina (arrowheads) (D) and loss of RGCs (arrowheads) (E and F). A bar plot (G) shows a significant decrease in the mean (SE) cell count of the RGC layer between rescued right eyes (WT-ND4 [triple Y-F capsid modifications] + G11778A) and mock-treated left eyes (green fluorescent protein + G11778A-ND4). INL indicates inner nuclear layer; ONL, outer nuclear layer. Scale bar in parts A and D indicates 200 µm; B and E, 100 µm; and C and F, 50 µm. ^aP = 6.3 × 10⁻¹⁶.

Figure 4. Histopathologic Findings of the Optic Nerve

A gross dissection specimen (A) revealed marked atrophy of the mock-treated left optic nerve (left arrowheads) along its entire length from the back of the left eye (OS) to the optic chiasm. Wild-type (WT) ND4 packaged with the triple Y-F capsid–modified (TM) self-complementary adeno-associated viral vector prevented atrophy of the right optic nerve (right arrowheads) in the right eye (OD) that was of normal caliber along its entire length. Schematic illustration (B) of part A. A bar plot (C) shows differences in optic nerve diameters between the right (WT-ND4[TM] + G11778A) and left (green fluorescent protein + G11778A-ND4) eyes were highly significant. Cross sections through the optic nerve taken approximately 1mmbehind the globe confirmed the marked atrophy of the left optic nerve (D). Cross section of the rescued right optic nerve shows no marked atrophy (E). Scale bar in parts D and E indicates 100 µm. ^aP = 1.3 × 10⁻¹².

Figure 5. Ultrastructural Findings in the Optic Nerve

Transmission electronmicrographs counterstained with uranyl acetate showed axonal loss with degenerating axon profiles (arrowheads) in mock-treated optic nerves even 1 year after intraocular injections (A); loss of axons and fibers with thin myelin lamellae and degenerating axons (arrowheads) were evident in mock-treated left eyes (B); and axons were numerous in rescued optic nerves, but some degenerating fibers were evident (arrowhead) (C). Scale bar in part A indicates 10 µm; B and C, 2 µm; and a indicates axon.

Figure 6. Retinal Photographs of Rhesus Macaques

Retinal fundus images of the right eye of a rhesus monkey before injection (A) of self-complementary adeno-associated viral vector–P1ND4v2 (AAV) and 5 days (B), 1 week (C), 2 weeks (D), 1 month (E), 2 months (F), and 3 months (G) after injection show that the macula and optic disc remained normal and similar to the uninjected left eye (H-N). The AAV-injected right eye of a second animal is normal at baseline (O), and at 5 days (P), 1 week (Q), and 2 weeks (R) after injection, but developed mild haze from vitritis at 1 month postinjection (S) that cleared at 2 months (T) to 3 months (U) after injection. The uninjected left eye of the same animal (control) was normal before injection of the opposite eye (V) and at all time points after injection (V through AB).

Figure 7. Ocular Histopathologic Findings in Rhesus Macaques

Hematoxylin-eosin–stained sections of the right eye that developed transient vitritis shows no inflammation in the vitreous or retina 3 months after AAV injection (A–B). The retina of the uninjected left eye is shown (C). The injected right optic nerve shows no evidence of inflammation or swelling (D). Green fluorescent protein (GFP) expression in a primate injected with the triple Y-F capsid–modified self-complementary adeno-associated viral vector–GFP. The GFP (E) is seen in Bm 3a–labeled (F) macular retinal ganglion cells (G).