Ex vivo gene therapy for the preservation of retinal and central nervous system structure and function in a canine model of CLN2 neuronal ceroid lipofuscinosis

Dissertation supervisor: Dr. Martin L. Katz.Includes vita.The neuronal ceroid lipofuscinoses (NCLs) are an inherited group of related lysosomal storage disorders. The NCLs are characterized in general by the accumulation of autofluorescent lysosomal storage material (ceroid-type lipofuscionosis) and progressive neurodegeneration. Age of onset and disease duration differs among the various forms of NCL, as each is caused by mutations in one of at least 13 different genes. Typically, the NCLs as a whole are inherited in an autosomal recessive manner, though at least one autosomal dominant form exists. Age of onset generally occurs sometime between early to late childhood, though adult onset NCL has been seen rarely. Progressive loss of neurological function is present among all forms, with progressive vision impairment also very common. Ultimately, following symptom progression, the NCLs result in premature death, with the specific age range being form dependent. In almost all cases death has been attributed to pronounced loss of neurological functions. The CLN2 form of NCL (previously designated as classical late-infantile NCL) results from mutations in the gene TPP1 (h11p15.5), which encodes the soluble lysosomal enzyme tripeptidyl peptidase-1 (TPP1). TPP1 is a member of the sedolisin family of serine-carboyxl peptidases and is active within the lysosome, where it cleaves N-terminal tripeptides. A long-haired miniature Dachshund model of CLN2 was developed and is currently being maintained and studied at the University of Missouri Columbia in an effort to better understand and develop therapies for CLN2. Affected dogs are homozygous for a single base pair deletion within exon 4 of TPP1 (c.325delC) that alters the reading frame of the predicted mRNA after codon 107 and creates a premature stop codon at position 114. Ultimately, this mutation results in no functional enzyme being produced. TPP1 dogs develop symptom progression similar to that seen in human CLN2, with ataxia, cognitive changes coinciding with cerebral atrophy, and myoclonic seizures beginning at 7-8 months of age and progressively worsening through disease end-stage by 10-11 months at which time the dogs are euthanized. Additionally, affected dogs develop vision loss that progresses to blindness with corresponding progressive retinal degeneration, electroretinogram (ERG) deficits, and the development of focal retinal detachment lesions (retinopathy). The marked similarity in disease presentation between canine and human CLN2 as well as the overall resemblance between the canine and human nervous system and retina makes these dogs an ideal model for development and evaluation of putative therapies for eventual translation to human clinical trials. Previous studies conducted by the lab demonstrated that TPP1 enzyme replacement therapy delivered to the CNS via cerebrospinal fluid infusion is effective in ameliorating neurological symptoms, prolonging life, and reducing the amount of lysosomal storage material within the brain; however, this therapy did not result in the preservation of the retina. As a result, a more directed retinal therapy is necessary to prevent retinal degeneration and preserve vision. Studies were conducted utilizing a novel autologous ex vivo gene therapy approach, in which mesenchymal stem cells (MSCs) were enriched from bone marrow aspirates, transduced with viral vectors directing the constituent overexpression of TPP1, and injected intravitreally as a means of providing sustained TPP1 to the retina. Dogs were evaluated via ERG and scanning laser ophthalmoloscopic (SLO) and optical coherence tomography (OCT) imaging to gauge the effectiveness of the treatment in preserving the various retinal layers and preventing the development of focal detachment lesions. The treatment resulted in preservation of retinal structure and function that appeared dosedependent, with ERG responses remaining normal-like longer in dogs that received more cells. A similar trend was observed with regards to the CLN2-related changes in retinal structure. Regardless of dosage, the retinas of the treated eyes did eventually begin to degenerate, but at a much slower pace versus those of the contralateral control eyes. Further studies are necessary, and are in progress, to determine whether the ex vivo gene therapy approach is capable of preserving the retina beyond normal end-stage in the canine CLN2 model and if repeated therapeutic application has a beneficial effect relative to a single pre-symptomatic injection. Preliminary studies indicate that introduction of the TPP1-expressing MSCs into the cerebrospinal fluid is safe and slows central nervous system degeneration.Includes bibliographical references (pages 108-113)