Successful Gene Therapy Research Articles

Co-assembled Ca2+ Alginate-Sulfate Nanoparticles for Intracellular Plasmid DNA Delivery

Successful gene therapy requires the development of suitable carriers for the selective and efficient delivery of genes to specific target cells, with minimal toxicity. In this work, we present a non-viral vector for gene delivery composed of biocompatible materials, CaCl2, plasmid DNA and the semi-synthetic anionic biopolymer alginate sulfate (AlgS), which spontaneously co-assembled to form nanoparticles (NPs). The NPs were characterized with a slightly anionic surface charge (Zeta potential [ζ] = −14 mV), an average size of 270 nm, and their suspension was stable for several days with no aggregation. X-ray photoelectron spectroscopy (XPS) validated their ternary composition, and it elucidated the molecular interactions among Ca2+, the plasmid DNA, and the AlgS. Efficient cellular uptake (>80%), associated with potent GFP gene expression (22%–35%), was observed across multiple cell types: primary rat neonatal cardiac fibroblasts, human breast cancer cell line, and human hepatocellular carcinoma cells. The uptake mechanism of the NPs was studied using imaging flow cytometry and shown to be via active, clathrin-mediated endocytosis, as chemical inhibition of this pathway significantly reduced EGFP expression. The NPs were cytocompatible and did not activate the T lymphocytes in human peripheral blood mononuclear cells. Proof of concept for the efficacy of these NPs as a carrier in cancer gene therapy was demonstrated for Diphtheria Toxin Fragment A (DT-A), resulting in abrogation of protein synthesis and cell death in the human breast cancer cell line. Collectively, our results show that the developed AlgS-Ca2+-plasmid DNA (pDNA) NPs may be used as an effective non-viral carrier for pDNA.

Gene replacement therapy in a model of Charcot-Marie-Tooth 4C neuropathy.

Charcot-Marie-Tooth disease type 4C is the most common recessively inherited demyelinating neuropathy that results from loss of function mutations in the SH3TC2 gene. Sh3tc2-/- mice represent a well characterized disease model developing early onset progressive peripheral neuropathy with hypo- and demyelination, slowing of nerve conduction velocities and disturbed nodal architecture. The aim of this project was to develop a gene replacement therapy for treating Charcot-Marie-Tooth disease type 4C to rescue the phenotype of the Sh3tc2-/- mouse model. We generated a lentiviral vector LV-Mpz.SH3TC2.myc to drive expression of the human SH3TC2 cDNA under the control of the Mpz promoter specifically in myelinating Schwann cells. The vector was delivered into 3-week-old Sh3tc2-/- mice by lumbar intrathecal injection and gene expression was assessed 4-8 weeks after injection. Immunofluorescence analysis showed presence of myc-tagged human SH3TC2 in sciatic nerves and lumbar roots in the perinuclear cytoplasm of a subset of Schwann cells, in a dotted pattern co-localizing with physiologically interacting protein Rab11. Quantitative PCR analysis confirmed SH3TC2 mRNA expression in different peripheral nervous system tissues. A treatment trial was initiated in 3 weeks old randomized Sh3tc2-/- littermate mice which received either the full or mock (LV-Mpz.Egfp) vector. Behavioural analysis 8 weeks after injection showed improved motor performance in rotarod and foot grip tests in treated Sh3tc2-/- mice compared to mock vector-treated animals. Moreover, motor nerve conduction velocities were increased in treated Sh3tc2-/- mice. On a structural level, morphological analysis revealed significant improvement in g-ratios, myelin thickness, and ratios of demyelinated fibres in lumbar roots and sciatic nerves of treated Sh3tc2-/- mice. Finally, treated mice also showed improved nodal molecular architecture and reduction of blood neurofilament light levels, a clinically relevant biomarker for axonal injury/degeneration. This study provides a proof of principle for viral gene replacement therapy targeted to Schwann cells to treat Charcot-Marie-Tooth disease type 4C and potentially other similar demyelinating inherited neuropathies.

A Novel BaEVRless-Pseudotyped γ-Globin Lentiviral Vector Drives High and Stable Fetal Hemoglobin Expression and Improves Thalassemic Erythropoiesis In Vitro.

It has previously been demonstrated that the self-inactivating γ-globin lentiviral vector GGHI can significantly increase fetal hemoglobin (HbF) in erythroid cells from thalassemia patients and thus improve the disease phenotype in vitro. In the present study, the GGHI vector was improved further by incorporating novel enhancer elements and also pseudotyping it with the baboon endogenous virus envelope glycoprotein BaEVRless, which efficiently and specifically targets human CD34+ cells. We evaluated the hypothesis that the newly constructed vector designated as GGHI-mB-3D would increase hCD34+ cell tropism and thus transduction efficiency at low multiplicity of infection, leading to increased transgene expression. High and stable HbF expression was demonstrated in thalassemic cells for the resulting GGHI-mB-3D/BaEVRless vector, exhibiting increased transduction efficiency compared to the original GGHI-mB-3D/VSVG vector, with a concomitant 91% mean HbF increase at a mean vector copy number per cell of 0.86 and a mean transduction efficiency of 56.4%. Transduced populations also exhibited a trend toward late erythroid, orthochromatic differentiation and reduced apoptosis, a further indication of successful gene therapy treatment. Monitoring expression of ATG5, a key link between autophagy and apoptosis, it was established that this correction correlates with a reduction of enhanced autophagy activation, a typical feature of thalassemic polychromatophilic normoblasts. This work provides novel mechanistic insights into gene therapy-mediated correction of erythropoiesis and demonstrates the beneficial role of BaEVRless envelope glycoprotein compared to VSVG pseudotyping and of the novel GGHI-mB-3D/BaEVRless lentiviral vector for enhanced thalassemia gene therapy.

Transfection of the TRAIL gene into human mesenchymal stem cells using biocompatible polyethyleneimine carbon dots for cancer gene therapy

For effective gene therapy using Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL), which is a protein that selectively induces cancer cell apoptosis through human mesenchymal stem cells (hMSCs), low cytotoxicity branched polyethyleneimine (bPEI) 25k carbon dots (bPEI25k CDs) were investigated as a TRAIL–GFP gene (plasmid TRAIL–GFP, pTRAIL–GFP) carrier for delivery into hMSCs. bPEI25k CDs were prepared using one-step microwave-assisted pyrolysis, which has the advantage of low cytotoxicity and allows for bioimaging after gene delivery. The bPEI25k CDs had lower cytotoxicity levels at all tested concentrations, whereas the raw bPEI25k were cytotoxic in proportions up to a concentration by 25%. The complex containing bPEI25k CDs and pTRAIL–GFP was safely internalized and transfected into the hMSCs. It also revealed a cellular fluorescence imaging property induced by a unique characteristic of CDs. The amount of secreted TRAIL from bPEI25k CDs/pTRAIL–GFP@hMSCs was 25-fold higher than that from bPEI25k/pTRAIL–GFP@hMSCs, leading to a higher cytotoxic effect against the lung cancer cells. Therefore, bPEI25k CDs are recommended as an effective gene carrier for successful cancer gene therapy mediated through hMSCs.

FVIII expression by its native promoter sustains long-term correction avoiding immune response in hemophilic mice

AbstractHere we describe a successful gene therapy approach for hemophilia A (HA), using the natural F8 promoter (pF8) to direct gene replacement to factor VIII (FVIII)–secreting cells. The promoter sequence and the regulatory elements involved in the modulation of F8 expression are still poorly characterized and biased by the historical assumption that FVIII expression is mainly in hepatocytes. Bioinformatic analyses have highlighted an underestimated complexity in gene expression at this locus, suggesting an activation of pF8 in more cell types than those previously expected. C57Bl/6 mice injected with a lentiviral vector expressing green fluorescent protein (GFP) under the pF8 (lentiviral vector [LV].pF8.GFP) confirm the predominant GFP expression in liver sinusoidal endothelial cells, with a few positive cells detectable also in hematopoietic organs. Therapeutic gene delivery (LV.pF8.FVIII) in hemophilic C57/Bl6 and 129-Bl6 mice successfully corrected the bleeding phenotype, rescuing up to 25% FVIII activity, using a codon-optimized FVIII, with sustained activity for the duration of the experiment (1 year) without inhibitor formation. Of note, LV.pF8.FVIII delivery in FVIII-immunized HA mice resulted in the complete reversion of the inhibitor titer with the recovery of therapeutic FVIII activity. Depletion of regulatory T cells (Tregs) in LV-treated mice allowed the formation of anti-FVIII antibodies, indicating a role for Tregs in immune tolerance induction. The significant blood loss reduction observed in all LV.pF8.FVIII-treated mice 1 year after injection confirmed the achievement of a long-term phenotypic correction. Altogether, our results highlight the potency of pF8-driven transgene expression to correct the bleeding phenotype in HA, as well as potentially in other diseases in which an endothelial-specific expression is required.

Chronic granulomatous disease 2018: advances in pathophysiology and clinical management

Chronic granulomatous disease (CGD) is a rare immunodeficiency disorder of phagocytic cells resulting in failure to kill a characteristic spectrum of bacteria and fungi and to resolve inflammation. The last few years have witnessed major advances in pathogenesis and clinical management of the disease: Better understanding of 3 physiologic anti-inflammatory functions of NADPH oxidase-derived reactive oxygen species: Promotion of the clearance of dying host cells, suppression of inflammasomes, and regulation of type I interferon signalling. This insight is opening new avenues for targeted drug interventions. Advances in reduced intensity conditioning (RIC) for allogeneic hematopoietic stem cell transplantation (HSCT) make it a promising and safe procedure even for fragile patients with ongoing severe infection or hyperinflammation. Encouraging early data of a multicenter trial of gene-replacement therapy using a self-inactivated lentiviral vector. Combining targeted anti-infectious/anti-inflammatory measures and considering extended indications for curative HSCT are key to improving patient outcome further. Gene therapy will likely become a viable option for disease correction, but long-term assessment is not yet possible. Statement of novelty: We discuss important advances in pathogenesis and treatment of CGD that will change our approach to clinical management.

Basic and Clinical Application of Adeno-Associated Virus-Mediated Genome Editing.

Traditional gene therapy (gene replacement) has made a breakthrough in treating inherited diseases. Adeno-associated virus (AAV) has emerged as a highly promising vector with innate ability, boosting the development of gene replacement and gene targeting. With the recent advance of engineered nucleases that work efficiently in human cells, AAV mediated-genome editing with nucleases has raised hopes for in situ gene therapy of inherited and non-inherited diseases. Here, the applications of AAV-mediated genome editing are highlighted, and the prospect of AAV and nucleases that will render extension of such success in clinical gene therapy is discussed.

Transcriptome Profiling and Molecular Therapeutic Advances in Cystic Fibrosis: Recent Insights.

In cystic fibrosis (CF), mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene disrupt the capacity of the encoded protein to function as a channel to transport chloride ions and water across cell membranes. The consequences are deleterious, system-wide, and immensely variable, even among patients with the same CFTR genotype. This underscores the need to characterize the mechanisms contributing to CF pathophysiology. Gene replacement and gene editing therapies have been pursued intensively and are expected to provide a one-time treatment for CF. However, gene replacement therapy is limited by the lack of efficient vectors to deliver functional copies of CFTR to cells without immunological complications, while gene editing technologies such as CRISPR/Cas9 are still in their infancy, mainly useful in somatic cells and limited by off-target insertions. Small molecule treatments targeted at potentiating or correcting CFTR have shown clinical benefits, but they are limited to a few CFTR mutations and insufficient to overcome challenges related to clinical heterogeneity. Transcriptome profiling approaches have emerged as robust tools capable of characterizing phenotypic variability and revealing novel molecular targets with therapeutic potential for CF. We summarize current insights gained through transcriptome profiling approaches in CF studies and recent advances in molecular therapeutics.

Value-Based Pricing for Emerging Gene Therapies: The Economic Case for a Higher Cost-Effectiveness Threshold.

While one-time gene replacement therapies may offer transformative innovation for the management of ultrarare, health-catastrophic diseases, they also pose challenges to the current U.S. health care system. Historically, the United States and other countries have demonstrated a willingness to support higher prices for health gains in rare diseases. However, payers may be ill-prepared to address reimbursement based on single administrations associated with gene therapies. As yet, there is no consensus on how to appropriately reward gene therapy innovation. The purpose of this article is to characterize challenges for traditional approaches to assessing the value of one-time gene replacement therapies and to provide a health economic rationale for a higher value-based cost-effectiveness threshold (CET). There is a general recognition that ultrarare, health-catastrophic conditions should be judged against a higher CET. The Institute for Clinical and Economic Review in the United States has discussed a range of up to $500K per quality-adjusted life-year (QALY) gained for ultrarare diseases, and the National Institute for Health and Care Excellence in the United Kingdom has described a variable threshold up to £300,000 per QALY depending on the magnitude of the health gains. In practice, health technology assessment decision makers often make comparisons to "benchmarks" to justify both standard and extraordinary CETs. We briefly review and present a list of relevant benchmarks. We also sketch out how a broader concept of value could provide the basis for higher CETs for some ultrarare diseases. This approach is outlined by the recent International Society for Pharmacoeconomics and Outcomes Research Special Task Force on Value Assessment Frameworks. In addition to the QALY gains, other elements of value related to uncertainty may also be important. They include insurance value, severity of disease, real option value, value of hope, and equity. A gene therapy currently in development for the treatment of spinal muscular atrophy (SMA) provides an exemplar for discussing the issues that accompany one-time gene replacement therapies. It is imperative that we find a consensus on how to appropriately reward value created by these gene therapies to incentivize appropriate risk taking and investments by their developers-a higher CET would, by economic logic, support a higher value-based price. If consensus on appropriate rewards cannot be found for safe and effective gene therapies for diseases such as SMA with clear criticality and unmet need, it will be even more difficult to do so for diseases where the value provided is less apparent. DISCLOSURES: Funding for the writing of this article was provided by AveXis Pharmaceuticals, which reviewed the manuscript and contributed feedback during manuscript development. The authors had final editorial control. Jackson and Paul are employees of MME, a biopharmaceutical consulting firm that received funding from AveXis for work on this project. Jackson and Paul also report consulting fees from numerous other biopharmaceutical companies outside of this project. Garrison reports consulting fees from AveXis for work on this project and advisory/consultancy fees from BioMarin, Roche, Novartis, and Pfizer unrelated to this project. Kenston is a former employee of AveXis and reports consulting fees from AveXis for this project and for other projects outside of this work.

Improved\xa0TMC1 gene therapy restores hearing and balance in mice with genetic inner ear disorders

Fifty percent of inner ear disorders are caused by genetic mutations. To develop treatments for genetic inner ear disorders, we designed gene replacement therapies using synthetic adeno-associated viral vectors to deliver the coding sequence for Transmembrane Channel-Like (Tmc) 1 or 2 into sensory hair cells of mice with hearing and balance deficits due to mutations in Tmc1 and closely related Tmc2. Here we report restoration of function in inner and outer hair cells, enhanced hair cell survival, restoration of cochlear and vestibular function, restoration of neural responses in auditory cortex and recovery of behavioral responses to auditory and vestibular stimulation. Secondarily, we find that inner ear Tmc gene therapy restores breeding efficiency, litter survival and normal growth rates in mouse models of genetic inner ear dysfunction. Although challenges remain, the data suggest that Tmc gene therapy may be well suited for further development and perhaps translation to clinical application.

Cost-effectiveness analysis of using onasemnogene abeparvocec (AVXS-101) in spinal muscular atrophy type 1 patients

ABSTRACTBackground: Spinal muscular atrophy type 1 (SMA1) is a devastating genetic disease for which gene-replacement therapy may bring substantial survival and quality of life benefits.Objective: This study investigated the cost-effectiveness of onasemnogene abeparvovec (AVXS-101) gene-replacement therapy for SMA1.Study design: A Markov model was used to estimate the incremental cost-effectiveness ratio (ICER), expressed as cost/quality-adjusted life year ($/QALY), of AVXS-101 versus nusinersen over a lifetime. Survival, healthcare costs and QALYs were estimated using natural history data for SMA patients who achieved motor milestones (sitting/walking). Health utility weights were obtained from the CHERISH trial.Setting: USA; commercial payer perspectiveParticipants: SMA1 infantsInterventions: AVXS-101 was compared to nusinersen.Main outcome measure: The primary outcome was the ICER.Results: Expected survival (undiscounted) over a lifetime predicted by the model was 37.20 life years for AVXS-101 and 9.68 for nusinersen (discounted QALYs, 15.65 and 5.29, respectively). Using a potential AVXS-101 price range ($2.5-5.0M/treatment), the average lifetime cost/patient was $4.2–6.6M for AVXS-101 and $6.3M for nusinersen. The ICER range was (-$203,072) to $31,379 per QALY gained for AVXS-101 versus nusinersen, indicating that AVXS-101 was cost-effective with prices of ≤$5M.Conclusion: Single-dose AVXS-101 was cost-effective compared to chronic nusinersen for SMA1 patients.

Chinese Medicine Protein and Peptide in Gene and Cell Therapy.

The success of gene and cell therapy in clinic during the past two decades as well as our expanding ability to manipulate these biomaterials are leading to new therapeutic options for a wide range of inherited and acquired diseases. Combining conventional therapies with this emerging field is a promising strategy to treat those previously-thought untreatable diseases. Traditional Chinese medicine (TCM) has evolved for thousands of years in China and still plays an important role in human health. As part of the active ingredients of TCM, proteins and peptides have attracted long-term enthusiasm of researchers. More recently, they have been utilized in gene and cell therapy, resulting in promising novel strategies to treat both cancer and non-cancer diseases. This manuscript presents a critical review on this field, accompanied with perspectives on the challenges and new directions for future research in this emerging frontier.

Surgical Methods for Inner Ear Gene Delivery in Neonatal Mouse.

Inner ear gene therapy offers great potential as a treatment for hearing loss and dizziness. The surgical method used to deliver gene therapy into the inner ear is a critical step in determining the success of inner ear gene therapy. Here we describe two commonly used surgical methods for gene delivery in neonatal mouse inner ear: the round window approach and the posterior semicircular canal approach. Both of these approaches are effective at delivering gene therapy to the neonatal mouse inner ear.

Connexin-43 Enhances the Redesigned Cytosine Deaminase Activity for Suicide Gene Therapy in Human Breast Cancer Cells.

Background:Escherichia coli cytosine deaminase (CD) converts 5-fluorocytosine (5-FC), a prodrug, into 5-fluorouracil (5-FU), a chemotherapeutic drug. However, the poor binding affinity of CD towards 5-FC as compared to the natural substrate cytosine, limits its application towards a successful suicide gene therapy. Although F186W mutant was developed to enhance the effect of wild-type CD, still scope for its improvement remains to further minimize the dose-dependent cytotoxicity of the drugs. Hence, in this study, we employ the anti-tumour attribute of the gap junction forming protein connexin-43 (Cx43) in conjunction with CD or F186W mutant.Methods:Lipofectamine was used to co-transfect CD/F186W-pVITRO2 and Cx43-pEGFP-N1 plasmids construct into MCF-7 cells. Comparative analysis of cell viability was observed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) and trypan blue–based assays. To further confirm the mode of cell death was apoptosis, propidium iodide and annexin V/7-aminoactinomycin D (7-AAD)-based apoptosis assays were performed.Results:Semi-quantitative polymerase chain reaction (PCR) confirmed the expression of both Cx43 and CD/F186W genes after transfection. Furthermore, cell viability assays revealed the enhanced activity of F186W-Cx43 compared with CD-Cx43 and F186W alone. The trend of the reduction in cell viability was also reflected in the flow cytometry–based apoptosis analyses. Overall, F186W-Cx43 combination demonstrated its superiority over the CD-Cx43 and F186W mutant alone.Conclusions:The enhanced cytotoxic activity of F186W mutant was further amplified by gap junction protein Cx43.

Generation of Novel Cardiac Specific AAV Vectors by Directed Evolution in Human iPSC Derived Cardiomyocytes

Recombinant adeno-associated viral (rAAV) vectors have emerged as one of the most promising gene therapy vectors. However, recent evidence has indicated that successful rAAV-mediated gene therapy in animal models may not translate to the same therapeutic benefit in humans. This is mainly due to the species difference in rAAV transduction efficiency, thus leading to the search for new capsids which exhibit high efficiency in human cells. This may be achieved through capsid shuffling, in which rAAV capsid gene sequences can be reconstituted from fragments of naturally occurring rAAV capsids to generate a library comprised of novel rAAVs with unique capsid protein sequences. This library could be screened using Directed Evolution (DE) in human cardiomyoctyes to select for cardiotropic rAAVs. In the development of strategies for personalised medicine, patient-specific iPSC-CMs could be used to evolve patent specific rAAVs. To test this strategy, we have used an rAAV library (rAAV1-12) to perform DE in human iPSC-CMs. The resulting rAAVs were then recovered from cell lysates and used for subsequent rounds of selection in fresh iPSC-CMs. After six rounds of selection, the recovered rAAVs were analysed for enrichment of cardiotropic candidates. Five candidates emerged. Functional analysis using rAAV-GFP indicated that while rAAV6 was the most efficient at transducing iPSC-CM among natural rAAVs, two of our novel variants led to higher GFP expression at the same vector dose. Our results validate the use of DE to develop novel, highly functional rAAVs for use in clinical studies.

Lysosomal diseases: Overview on current diagnosis and treatment.

Lysosomal diseases (LDs), also known as lysosomal storage diseases (LSDs), are a heterogeneous group of conditions caused by defects in lysosomal function. LDs may result from deficiency of lysosomal hydrolases, membrane-associated transporters or other non-enzymatic proteins. Interest in the LD field is growing each year, as more conditions are, or will soon be treatable. In this article, we review the diagnosis of LDs, from clinical suspicion and screening tests to the identification of enzyme or protein deficiencies and molecular genetic diagnosis. We also cover the treatment approaches that are currently available or in development, including hematopoietic stem cell transplantation, enzyme replacement therapy, small molecules, and gene therapy.

Basic Concepts in Viral Vector-Mediated Gene Therapy.

Today any researcher with the desire can easily purchase a viral vector. However, despite the availability of viral vectors themselves, the requisite knowledge that is absolutely essential to conducting a gene therapy experiment remains somewhat obscure and esoteric. To utilize viral vectors to their full potential, a large number of decisions must be made, in some instances prior to even obtaining the vector itself. For example, critical decisions include selection of the proper virus, selection of the proper expression cassette, whether to produce or purchase a viral vector, proper viral handling and storage, the most appropriate delivery method, selecting the proper controls, how to ensure your virus is expressing properly, and many other complex decisions that are essential to performing a successful gene therapy experiment. The need to make so many important decisions can be overwhelming and potentially prohibitive, especially to the novice gene therapist. In order to aid in this challenging process, here we provide an overview of basic gene therapy modalities and a decision tree that can be used to make oneself aware of the options available to the beginning gene therapist. This information can be used as a road map to help navigate the complex and perhaps confusing process of designing a successful gene therapy experiment.

Gene Delivery Using Lipoplexes and Polyplexes: Principles, Limitations and Solutions.

Gene therapy has attracted considerable attention for the treatment of genetic and acquired diseases. Successful gene therapy occurs when the therapeutic genes penetrate targeted cells and become available to the intracellular active site. Currently, a promising approach in gene delivery is the use of nonviral gene delivery vectors that lack immunogenicity but have low toxicity and potential tissue specificity. The widely used, existing nonviral gene vectors are cationic lipids and polymers that can pass across extracellular and intracellular barriers. However, the toxicity of these vectors is a barrier to their use. Currently, the disadvantages of nonviral vectors have been minimized by several modifications. The main purpose of this review is to describe the pros and cons of gene delivery using cationic lipids and polymers.

Onasemnogene abeparvovec. AAV vector-based gene-replacement therapy, Treatment of spinal muscular atrophy

Onasemnogene abeparvovec. AAV vector-based gene-replacement therapy, Treatment of spinal muscular atrophy

Time Course of Disease Progression of PRPF31-mediated Retinitis Pigmentosa

Variants in PRPF31, a splicing factor, are a common cause of autosomal dominant retinitis pigmentosa (RP). Deleterious variants are thought to cause disease by haploinsufficiency. In anticipation of upcoming replacement gene therapy trials, we present the phenotype and clinical progression of a large cohort of patients with PRPF31-mediated RP. Cross-sectional with retrospective review. A total of 26 patients with RP and 5 asymptomatic individuals, all with deleterious variants in PRPF31 (from 13 families), were selected from our database of patients followed longitudinally. Ages ranged from 9 to 77 years (mean 47 years), with an average follow-up time of 16 years. All patients underwent ophthalmic examination including psychophysical tests, electrophysiology, and imaging. All available records were reviewed retrospectively. Additionally, all patients were contacted, and all available patients (n= 7) were examined in an additional prospective follow-up visit. Age of onset ranged from 6 to 71 years, without apparent relationship to specific variant. Two adults (aged 42 and 77 years) and 3 teenaged children were found to harbor a mutation with no evidence of RP. In those with RP, visual field area (spot size III) declined exponentially at a rate of 8.1% per year of disease duration (P < .001, 95% confidence interval [CI] 5.6-10.6), cone electroretinogram amplitude declined exponentially at a rate of 7.3% per year of disease duration (P < .001, 95% CI 5.4-9.1), and ellipsoid zone area declined exponentially at a rate of 5.4% per year of disease duration (P<.001, 95% CI 3.7-7.1). PRPF31-mediated retinitis pigmentosa is characterized by a variable age of onset. Once disease develops, it follows a predictable exponential time course.