Nightstar Therapeutics R&D

Maintain & Restore Vision

Nightstar Therapeutics R&D Day September 24, 2018

Email your questions to: [email protected]

* This presentation is intended for investor relations purposes only and is not intended for any other purpose, including the promotion of Nightstar’s product candidates. Disclaimers

This presentation contains “forward-looking statements” within the meaning of the Private Securities Litigation Reform Act of 1995. The words “believe,” “anticipate,” “could,” “intend,” “estimate,” “will,” “would,” “may,” “should,” “project,” “target,” “track,” “expect” or other similar expressions are intended to identify forward-looking statements, although not all forward-looking statements contain these identifying words. All statements contained in this presentation other than statements of historical facts are forward-looking statements, including, without limitation: our planned and ongoing clinical trials for NSR-REP1 and NSR-RPGR, including our Phase 3 STAR trial in choroideremia and Phase 1/2 XIRIUS trial in X-linked retinitis pigmentosa; potential results and timelines relating to the dose escalation study in the XIRIUS trial and the planned expansion study in the XIRIUS trial; the potential utility of prior preclinical and clinical data and the data and endpoints presented herein in predicting future clinical results for our product candidates and any results of assessments to be conducted by regulatory agencies; the doses of NSR-RPGR to be used in the expansion study in the XIRIUS trial and future trials of NSR-RPGR; the continued clinical development of our pipeline; the timelines associated with our research and development programs including the timing of patient enrollment and the release of data from ongoing clinical trials and studies; the prevalence of patient populations for our targeted indications; and statements about our cash position and sufficiency of capital resources to fund our operating requirements, trends and other factors that may affect our financial results. These forward-looking statements are based on management's current expectations of future events as of the date of this presentation and are subject to a number of involve substantial known and unknown risks, uncertainties and other factors that may cause our actual results, levels of activity, performance or achievements to be materially different from the information expressed or implied by these forward-looking statements, including those related to the timing and costs involved in commercializing any product candidate that receives regulatory approval; the initiation, timing and conduct of clinical trials; the availability of data from clinical trials and expectations for regulatory submissions and approvals; whether interim results of a clinical trial will be predictive of the final results of the trial; whether results of small or early stage clinical trials will be predictive of the results of later-stage trials; our scientific approach and general development progress; the availability or commercial potential of the our product candidates; the sufficiency of our cash resources; and other risks and uncertainties set forth in Item 3.D. "Risk Factors" section of our Annual Report on Form 20-F for the year ended December 31, 2017 and subsequent reports that we file with the U.S. Securities and Exchange Commission. We may not actually achieve the plans, intentions, estimates or expectations disclosed in our forward-looking statements, and you should not place undue reliance on our forward-looking statements. Actual results or events could differ materially from the plans, intentions, estimates and expectations disclosed in the forward-looking statements we make. We anticipate that subsequent events and developments will cause our views to change. We are under no duty to update any of these forward-looking statements after the date of this presentation to conform these statements to actual results or revised expectations, except as required by law. You should, therefore, not rely on these forward-looking statements as representing our views as of any date subsequent to the date of this presentation. Any reference to our website address in this presentation is intended to be an inactive textual reference only and not an active hyperlink. 2 Agenda

8:00 – 8:10 AM Nightstar’s Mission Dave Fellows, Chief Executive Officer, Nightstar Therapeutics 8:10 – 8:20 AM X-Linked Retinitis Pigmentosa (XLRP) – Byron L. Lam, M.D., Greene Professor of Ophthalmology, Bascom Background and Efficacy Endpoints Palmer Eye Institute, University of Miami School of Medicine 8:20 – 8:50 AM XIRIUS Phase 1/2 Trial: Dose Escalation Study Robert MacLaren, MBChB, D.Phil., FACS, Professor of Preliminary Results Ophthalmology, University of Oxford; Scientific Founder, Nightstar Therapeutics 8:50 – 9:05 AM Q&A 9:05 – 9:20 AM NSR-REP1 for the Treatment of Tuyen Ong, M.D., MRCOphth, MBA, Chief Development Officer, Choroideremia Nightstar Therapeutics Robert MacLaren, MBChB, D.Phil., FACS, Professor of Ophthalmology, University of Oxford; Scientific Founder, Nightstar Therapeutics 9:20 – 9:30 AM Hybrid CMC Model in Gene Therapy Julian Hanak, Senior Vice President, Global Head of CMC, Nightstar Therapeutics 9:30 – 9:35 AM NSR-ABCA4 for the Treatment of Stargardt Robert MacLaren, MBChB, D.Phil., FACS, Professor of Disease Ophthalmology, University of Oxford; Scientific Founder, Nightstar Therapeutics 9:35 – 9:45 AM Q&A 9:45 – 10:00 AM Closing Remarks Dave Fellows, Chief Executive Officer, Nightstar Therapeutics 3 Our Mission: David Fellows Maintain and Restore Chief Executive Officer, Vision in Blinding Nightstar Therapeutics Inherited Retinal 8:00 AM to 8:10 AM Diseases Building the Leading Retinal Gene Therapy Company

Phase 3 Registrational Trial in Choroideremia (CHM) Ongoing (RMAT June 2018)

Durable Treatment Effect Observed in CHM Phase 1/2 Trial (2+ years)

XLRP Phase 1/2 Preliminary Proof of Concept Safety and Efficacy Data

Multiple Gene Therapy Pipeline Programs from Oxford University

Sizeable Markets with High Unmet Medical Need

Hybrid CMC Model: In-house Process Development & QA w/ Outsourced Facilities

5 Advanced Pipeline of Novel Gene Therapy Candidates Targeting Inherited Retinal Diseases

Prevalence Stage of Program Indication (US/EU5)1 Development Next Milestone 13K Complete Phase 3 NSR-REP1 Choroideremia Phase 3 (1:50K) Enrollment: 1H 2019

17K Initiation of Expansion NSR-RPGR X-linked Retinitis Pigmentosa Phase 1/2 (1:40K) Cohort: 4Q 2018

65K NSR-ABCA4 Stargardt Disease Preclinical (1:10K)

10K NSR-BEST1 Best Disease Preclinical (1:67K)

We retain worldwide commercial rights to all of our product candidates

1 Management estimates based on published epidemiology. 6 X-Linked Retinitis Byron L. Lam, M.D. Pigmentosa - Greene Professor of Ophthalmology, Bascom Palmer Eye Institute Background and Efficacy University of Miami School of Medicine Endpoints 8:10 AM to 8:20 AM X-Linked Retinitis Pigmentosa: Progressive Photoreceptor Degeneration that Leads to Blindness; No Treatment Options

• 10-20% of retinitis pigmentosa is X-linked1 ▪ 70% of XLRP caused by RPGR mutations XLRP Patient ▪ Mostly males but some females affected Peripheral “bone spicules” & retinal atrophy • Orphan Disease ~ 1:40,0002 ▪ 17,000 RPGR patients in U.S./EU5 • Median age of legal blindness is 45* ▪ Much younger than for other forms of RP • Disease progression4 ▪ Early – Nyctalopia (Night blindness) ▪ Mid - Peripheral vision field constriction ▪ Late - Central vision deterioration / loss • Effects can impact patient’s work, school and social interactions

Sources: (1) Birch, Retinal Physician, April 2018; (2) Management estimates based on published epidemiology; (*) Sandberg et al., Invest. Ophthalmol. Vis. Sci., March 2007; 8 (4) Iftikhar et al., Ophthal Retina, May 2018. (Image) Hamel, Orphanet Journal of Rare Diseases, October 2006 Pathology of Disease: RPGR Mutations Can Lead to Photoreceptor Death

• RPGR localized in cilium tract ▪ Connective body between inner and outer segment of photoreceptors

Basal Bodies • Connecting cilium used for active transport of proteins between inner and outer segments ▪ Controls bidirectional transport between two discrete compartments • RPGR mutations associated with abnormal protein

Source: Birch, Retinal Physician, April 2018. transport across cilium 9 Preservation of Photoreceptors: Early Versus Late Effects

Potential Early Effect Potential Late Effect Light Sensitive Retina Retinal Sensitivity: Microperimetry Retinal Structure: Ellipsoid Zone

Outer Nuclear Layer

Photoreceptor Cells

Inner and Outer Segments

Retinal Pigment Epithelium (RPE)

Bruch’s Membrane Choroid Treatment area Microperimetry grid

Sources: (1) Brightfocus (2) Iftikhar et al., Ophthal Retina, 2018 (3) adapted from Birch et al., JAMA Ophthalmology, Sept. 2013. 10 Potential Long-term Endpoint for XLRP EZ Width/Area: Structural Measurements of Degenerative Photoreceptor Loss

Ellipsoid Zone (EZ) RPE inner border BM / choroid

• EZ: photoreceptor inner/outer segment junction1 • In XLRP, studies have shown mean EZ decreases annually2: ▪ 7% (width) / 13% (area) / 0.86o / 248µm • Goal for therapy - reduction in the rate of photoreceptor loss: ▪ “The comparison should be made …with intervals of 6 months or more...” 3

Sources: (1) Staurenghi et al., Ophthalmology, Aug 2014; (2) adapted from Birch et al., JAMA Ophthalmology, Sept. 2013. (3) FDA draft guidance “Human Gene Therapy for Retinal Disorders” 11 published 7/12/18 Microperimetry (MP): Functional Assessment of Retinal Sensitivity & Potential Early Endpoint for XLRP

Example XLRP Patient Retinal Sensitivity Map

Video source: (Left) MAIA Microperimeter General Presentation Video; (Right) Retinal sensitivity map shown is an example of an image from an XLRP patient 12 Microperimetry: Functional Measurement of Macula Retinal Sensitivity Better Marker of Visual Function than Visual Acuity1 XLRP Patient Image Detailed Retinal Imaging • Realtime imaging of entire macula • Automated eye tracking MAIA Microperimetry • Stimulation of same retinal loci across tests/visits

Island(s) of Vision • Retinal sensitivity heatmap • 68 stimulus points • Increased accuracy & sensitivity

Scotoma(s) • Areas of retina unable to perceive any stimuli

Source: (1) Iftikhar et al., Ophthal Retina, 2018. Least Most 13 sensitivity sensitivity Microperimetry: High Sensitivity and Correlated with Vision-related Quality of Life Measurements

“For the RP patients, our results suggest enhanced defect detection by the MP-1 compared to the HFA.” 1

“These results indicate that macular sensitivity determined by MP1 is a good predictor of QOL in RP patients with relatively good vision.” 2

Sources: (1) Acton et al., Optom Vis Sci, July 2012. (2) Sugarawa et al., Jpn J Ophthalmol, Aug 2011. Abbreviations: MP – microperimetry; HFA – Humphrey Field Analyzer; VA – visual acuity; QoL – quality of life 14 Decreased Sensitivity Across 68 Loci Most Relevant in Early Disease Stages

Worsening

All examples shown are illustrative examples used for educational purposes and are not meant to demonstrate the potential safety or efficacy of any product Source: Iftikhar et al., Ophthal Retina, 2018. 15 Decreased Sensitivity Across Central 16 Loci Most Relevant with Decreasing Visual Field

Worsening

All examples shown are illustrative examples used for educational purposes and are not meant to demonstrate the potential safety or efficacy of any product Source: Iftikhar et al., Ophthal Retina, 2018. 17 Expectation in Untreated Retinitis Pigmentosa Patients: Microperimetry Retinal Sensitivity Decreases Over Time Mean Yearly Decline in Retinal Sensitivity (dB) 68 loci Central 16 Edge of scotoma Seeing retina

-0.4 dB -0.6 dB

-1.1 dB -1.3 dB

Source: Iftikhar et al., Ophthal Retina, 2018. Data from Nidek MP-1 using 68 loci pattern. 18 XIRIUS Phase 1/2 Trial: Robert MacLaren, Dose Escalation Study MBChB, D.Phil., FACS Professor of Ophthalmology, University of Oxford Preliminary Results Scientific Founder, Nightstar Therapeutics 8:20 AM – 8:50 AM RPGR is Alternatively Spliced

Intron 15 donor acceptor

5’ Exons 1-14 15 …GGGAGGAGAG 16 Exons 17-19 3’ RPGR primary RNA transcript GUGAGU AG

U1/6 sRNA NUCLEUS

nuclear membrane nuclear membrane CYTOPLASM

Ubiquitous RPGR mRNA

Source: Fischer et al., (2017) Codon-Optimized RPGR Improves Stability and Efficacy of AAV8 Gene Therapy in Two Mouse Models of X-Linked Retinitis Pigmentosa. Molecular Therapy.. 20 RPGR is Alternatively Spliced

Intron 15 donor acceptor

5’ Exons 1-14 15 …GGGAGGA GAG 16 Exons 17-19 3’

AG GUGAGU AG RPGR primary RNA transcript

U1/6 sRNA NUCLEUS nuclear membrane nuclear membrane CYTOPLASM

GUGAGU 3’ Photoreceptor specific RPGR mRNA - RPGRORF15

5’ Exons 1-14 15 16 Exons 17-19 3’ Ubiquitous RPGR mRNA

5’ Exons 1-14 15 …GGCAGGA 3’ AAV8.RPGR mRNA GUGAGU AG

U1/6 sRNA NUCLEUS nuclear membrane nuclear membrane CYTOPLASM

Truncated RPGR mRNA – potentially toxic Wu et al. A long-term efficacy study of gene replacement therapy for RPGR-associated retinal degeneration. Hum Molecular Genetics 2016

Codons AGU=Serine

5’ Exons 1-14 15 …GGCAG GA 3’ UCC=Serine AAV8.RPGR mRNA GUGUCC

U1/6 sRNA NUCLEUS

nuclear membrane nuclear membrane CYTOPLASM

Correct full-length RPGRORF15 mRNA from AAV8 vector

Dominik Fischer in Oxford

Western blot – RPGR bands corresponding to the full length protein

5’ Exons 1-14 15 …GGCAG GA 3’

GAA = Glu GAG = Glu GGA = Gly

GGG = Gly

Gly Gly Gly

Glu Glu NH2 Glu COOH RPGRORF15 translated into protein

Source: Fischer et al., (2017) Codon-Optimized RPGR Improves Stability and Efficacy of AAV8 Gene Therapy in Two Mouse Models of X-Linked Retinitis Pigmentosa. Molecular Therapy. 25

RPGR Glutamylation with TTLL5

Gly Gly

Gly TTLL5

Glu Glu Glu

RPGRORF15 post-translational modification of protein

inner segment outer segment

tubulin in photoreceptor cilium

Gly Gly Gly

Glu Glu Glu

RPGRORF15 post-translational glutamylation

Effect of RPGR ORF15 Deletion Gly

Gly TTLL5 Glu

RPGR with ORF15 deletion has reduced glutamylation

inner segment outer segment

tubulin in photoreceptor cilium

Gly Gly Glu

RPGRORF15 with reduced glutamylation due to deletion

inner segment outer segment

tubulin in photoreceptor cilium

Gly Gly Glu

RPGRORF15 with reduced glutamylation due to deletion

Sources: Fischer et al., (2017) Codon-Optimized RPGR Improves Stability and Efficacy of AAV8 Gene Therapy in Two Mouse Models of X-Linked Retinitis Pigmentosa. Molecular Therapy. Sun et al. Loss of RPGR glutamylation 30 underlies pathology of TTLL5 mutations. Proc Natl Acad Sci 2016. Effects of ORF15 Deletion

Source: Sun et al. Loss of RPGR glutamylation underlies pathology of TTLL5 mutations. Proc Natl Acad Sci 2016. 31 Codon Optimized NSR-RPGR In Vitro

Dr. Cristina Martinez Nuffield Laboratory of Ophthalmology, University of Oxford

Full length RPGR protein with no Full length and fully glutamylated splice variants in codon optimised ORF15 seen with GT335 immuno- RPGR construct (white arrow) staining in codon optimised RPGR CORRECT SPLICING CORRECT GLUTAMYLATION

Retinal sensitivity: Retinal Structure: Light Sensitive Retina1 Potential Early Effect Late Effect

Outer Nuclear Layer

Photoreceptor Cells

Inner and Outer Segments

Retinal Pigment Epithelium (RPE) • Utility as LT endpoint2 Bruch’s Membrane ▪ Choroid Treatment area Microperimetry Mean annual decrease in EZ width grid of 248 μm (7%)

Sources: (1) Brightfocus (2) Birch et al., JAMA Ophthalmology, Sept. 2013. Abbreviation: EZ – ellipsoid zone 33 XIRIUS: Phase 1/2 Clinical Trial Design Includes Two Studies

Dose Escalation Study Expansion Study

Follow-up Target Dose, (n=30) Data Visits Treatment Follow-up Monitoring Randomization (n=18 total*) Visits 2:1 allocation Committee Follow-up ratio Low-Dose Control, (n=15) Visits

Dose escalation

Genetically confirmed diagnosis of XLRP Eligibility Males 18 years and older Males 10 years and older Primary Safety: Incidence of dose limiting toxicities & Endpoint treatment emergent adverse events

Selected • Maintenance of vision (BCVA) Secondary • Changes in microperimetry Endpoints • Changes in SD-OCT Ellipsoid Zone Upcoming Q2 2019 – Six-month follow-up data expected Q4 2018 – Initiation of expansion study expected Milestones Q4 2019 – One-year follow-up data expected Mid 2019 – Preliminary data expected Note: FDA draft guidance “Human Gene Therapy for Retinal Disorders” published 7/12/18. * 6 dose cohorts of 3 patients each 34 Baseline Demographics: Generally Comparable Between Treated / Untreated Eyes • 18 patients enrolled: data available for 15 patients (cohorts 1-5) as of Sep 4, 2018 ▪ Data through varying time points available – up to 12 months for earlier cohorts ▪ Month 1 is common timepoint with full set of QC’d safety/efficacy data for cohorts 1-5 ▪ Month 1 data unavailable for cohort 6 – follow-up visits not completed

Cohort Dose # of Mean Baseline Mean Retinal Baseline Mean BCVA Baseline Mean EZ (gp) Pts Age Sensitivity (dB) (Letters) Width (microns) Treated Untreated Treated Untreated Treated Untreated Eye Eye Eye Eye Eye Eye 1 5 x 109 3 37 -0.2 -0.4 28 61 163 356 2 1 x 1010 3 34 +0.2 +1.5 50 57 1375 1384 3 5 x 1010 3 30 +0.2 +1.5 64 66 248 250 4 1 x 1011 3 33 +2.2 +2.3 64 63 462 792 5 2.5 x 1011 3 25 +8.9 +9.1 71 73 2411 3341

Note: Genetic profiles of patients across cohorts included 13 distinct genetic variants in various exons. 35 Safety Profile: NSR-RPGR Generally Well-Tolerated

• Current data from 15 treated patients (cohorts 1-5) indicate NSR-RPGR is generally well-tolerated ▪ No early discontinuations ▪ No dose limiting toxicity observed ▪ No serious treatment-related AEs • Mild transient drug-related inflammation in cohorts 4-5 • Independent DMC recommended escalation to maximum dose (5x1011)

Abbreviations: DMC – data monitoring committee; AE – adverse events 36 Goal of Treatment is to Maintain Function….

• Primary clinical goal is to maintain visual function ▪ 93% of treated eyes and 93% of untreated eyes (cohorts 1-5) maintained visual acuity through the latest follow-up ▪ No significant declines in ellipsoid zone or microperimetry sensitivity observed

• However …observation of a signal of efficacy was noted early in the study

37 Case Example: Durable Improvements In Both Area & Mean Sensitivity Seen at 1 Month and Sustained Through 6 Months

BASELINE MONTH 1

0.5 dB Treated 0.7 dB Untreated 3.4 dB Treated 0.5 dB Untreated MONTH 3 MONTH 6

6.6 dB Treated 0.5 dB Untreated 6.9 dB Treated 0.5 dB Untreated

Note: microperimetry images and data from responder in cohort 4. Baseline image shown reflects final image after triplicate testing. 38 Case Example: Durable Improvements In Both Area & Mean Sensitivity BASELINE

0.5 dB Treated 0.7 dB Untreated

Note: microperimetry images and data from responder in cohort 4. Baseline image shown reflects final image after triplicate testing. 39 Case Example: Durable Improvements In Both Area & Mean Sensitivity MONTH 1

3.4 dB Treated 0.5 dB Untreated

Note: microperimetry images and data from responder in cohort 4. 40 Case Example: Durable Improvements In Both Area & Mean Sensitivity MONTH 3

6.6 dB Treated 0.5 dB Untreated Note: microperimetry images and data from responder in cohort 4. 41 Case Example: Durable Improvements In Both Area & Mean Sensitivity MONTH 6

6.9 dB Treated 0.5 dB Untreated Note: microperimetry images and data from responder in cohort 4. 42 Proof-of-Concept Efficacy Summary

• Demonstrated proof of concept with durable dose-related improvements seen as early as month 1 across multiple microperimetry analyses

• Preliminary efficacy signals exhibited in cohorts 3-5 ▪ Across multiple microperimetry analyses ▪ Across multiple timepoints ⬧ MP data from latest available timepoints for cohorts 1-5 (ranging from 1 month in cohort 5 through 12 months in cohort 1) generally consistent with month 1 data presented below

• Inflammation may have dampened efficacy in higher dose cohorts 4 and 5 ▪ Steroids rescued efficacy dampened by inflammation

43 3/3 Patients in C3 and 2/6 in C4+C5 Exhibited Early Efficacy Signals

44 Overall Macula Sensitivity: Responders Seen in Dose Cohorts 3-5 (≥ 2 dB Increase)

Change in Mean Sensitivity (dB) at Month 1 Treated Untreated 5 1 responder 3 responders 4 1 responder 3 2.4 2dB Threshold 2 1.2 0.2 1 0.1 0.6 0.2 0.1 0 -0.1 -0.1 -1 -0.8 -2 Cohort 1 Cohort 2 Cohort 3 Cohort 4 Cohort 5 n=3 n=2 n=3 n=3 n=3

Note: The analysis is based on difference in mean sensitivities between baseline and one month follow-up. Triangle markers denote the range 45 of data (max and min). One patient in cohort 2 was excluded from the analysis because triplicate testing was not performed at baseline. Central 16 Sensitivity: Greater Efficacy Signal Seen (~ 6 dB in Cohort 3)

Change in Mean Sensitivity of Central 16 Loci (dB) at Month 1 9 Treated Untreated 7 6.1 5 3 0.5 1.2 1.4 1 0.4 -1 -0.2 -0.1 -0.8 -1.0 -0.6 -3 -5 Cohort 1 Cohort 2 Cohort 3 Cohort 4 Cohort 5 n=3 n=2 n=3 n=3 n=3

Note: The analysis is based on difference in mean sensitivities between baseline and one month follow-up. Triangle markers denote the range 46 of data (max and min). One patient in cohort 2 was excluded from the analysis because triplicate testing was not performed at baseline. Number of Improved Loci: Responders in Cohorts 3-5 (≥ 5 dB Improvement at 10% of Loci)

# of Patients with 5 dB Improvement at 10% Loci at Month 1

Treated Untreated 3

1 1 1 1

0 0 0 0 0

Cohort 1 Cohort 2 Cohort 3 Cohort 4 Cohort 5 n=3 n=2 n=3 n=3 n=3

Note: The analysis is based on difference in mean sensitivities between baseline and one month follow-up. One patient in cohort 2 was excluded from the analysis because triplicate testing was not performed at baseline. 47 Inflammation Dampened Efficacy in C4-C5 Treated Eyes…

Responder Criteria: Patients with 5 dB Improvement at 10% Loci

3 Inflammation May Have Dampened Efficacy

Lower Dose & More Severe Patients 1 1 1

Cohort 1 Cohort 2 Cohort 3 Cohort 4 Cohort 5 n=3 n=2 n=3 n=3 n=3

Note: The analysis is based on difference in mean sensitivities between baseline and one month follow-up for treated eyes. One patient in cohort 2 was excluded from the analysis because triplicate testing was not performed at baseline. 48 Case Example: Steroids Rescued Efficacy Dampened by Inflammation 8 Treated Untreated

6 Interim visit due to inflammation 4 Treatment with oral steroids

Mean Retinal Sensitivity (dB) Sensitivity Retinal Mean 0 Baseline Month 1 Week 5 Month 3 Month 6 Months Following NSR-RPGR Administration

Sensitivity Map for Treated Eye

Note: microperimetry data from responder in cohort 4. Microperimetry data for untreated eye at week five not available. 49 XIRIUS Trial Robert MacLaren, MBChB, D.Phil., FACS Dose Escalation Study: Professor of Ophthalmology, University of Oxford Review of Cohort 3 Data Scientific Founder, Nightstar Therapeutics 9:15 AM – 9:20 AM Consistency of Results Observed Across Different Efficacy Analyses in Cohort 3

Treated Untreated 100%

6.1

2.4

-0.1 -1.0 Change in Mean Sensitivity Change in Mean Sensitivity of Patients with 5 dB (dB) Central 16 Loci (dB) Improvement at 10% Loci

Note: The analysis is based on difference in mean sensitivities between baseline and one month follow-up. Triangle markers denote the range of data (max and min). 51 Overall Mean Sensitivity in Treated Eyes Improved Compared to Untreated to Untreated Eyes in Cohort 3

Treated Eye Untreated Eye 5 (n=3) (n=3) 4 3

2 2.4 2.4 2.3 1 0 0.0 -1 -0.3 -0.1 -0.5

-2 Change in Mean Retinal (dB) Sensitivity Retinal Mean in Change -3 Months Following NSR-RPGR Administration Baseline Month 1 Month 3 Month 6

Note: The analysis is based on difference in sensitivities between baseline and six month follow-up. 52 Triangle markers denote the range of data (max and min). Central 16 Mean Sensitivity in Treated Eyes Improved by ≥6 dB Compared to Untreated Eyes in Cohort 3

Treated Eye Untreated Eye 10 (n=3) (n=3)

6 6.1 5.6 4 5.1

2 Central Loci 16 Central 0

-2 -1.0 -1.1 -1.2 Change from BaselineSensitivity Mean in from Change -4 Months Following NSR-RPGR Administration Baseline Month 1 Month 3 Month 6

Note: The analysis is based on difference in sensitivities between baseline and six month follow-up. Triangle markers denote the range of data (max and min). 53 ≥5 dB Improvement in Retinal Sensitivity in More than 10% of Loci in Treated Eyes But Not Untreated Eyes in Cohort 3

Treated Eye Untreated Eye (n=3) (n=3) 100% 100% 100% 100% 80%

60%

40% Change Loci of 10% in Change

20% % Patients Minimum with dB 5 of Patients % 0% 0% 0% 0% Baseline Month 1 Month 3 Month 6 Months Following NSR-RPGR Administration

Note: The analysis is based on difference in sensitivities between baseline and six month follow-up. 54 NSR-RPGR Treated Eyes: Increases in Retinal Sensitivity Generally Sustained Across Various Thresholds

Cohort 3: Mean # of Loci with Increase in Retinal Sensitivity from Baseline

30 Month 1 Month 6 27 27 26 Treated Untreated 24 22 20 19 16 15 13 13 13 12 10 9 8 7 6 5 4 3 2 1 1 1 1 0

≥1 dB ≥2 dB ≥3 dB ≥4 dB ≥5 dB ≥6 dB ≥7 dB >=1 db >=2 db >=3 db >=4 db >=5 db >=6 db >=7 db

55 Untreated Eyes: More Loci With Decreases in Retinal Sensitivity

Cohort 3: Mean # of Loci with Decrease in Retinal Sensitivity from Baseline

Month 1 Month 6 13 12 11 Treated Untreated 12

9 8 8 7 6 5 4 3 3 2 1 1 1 1 1 1 0 0 0 0 0 0 0 0

>=1 db >=2 db >=3 db >=4 db >=5 db >=6 db >=7 db ≥1 db ≥2 db ≥3 db ≥4 db ≥5 db ≥6 db ≥7 db

56 All Treated Eyes in Cohort 3 Showed Improvement in Retinal Sensitivity

Treated Eyes Untreated Eyes

Baseline

Month One Month Month Six Month

C3-1 C3-2 C3-3 C3-1 C3-2 C3-3 Note: Baseline images shown reflect triplicate testing. 57 NSR-RPGR Treated Eyes Showed Early Improvements at Month 1 with Sustained Effects to Month 6

Treated Eyes Untreated Eyes Month Six Month One Month Six Month C3-1 C3-2 C3-3 C3-1 C3-2 C3-3 Legend: No Change ≥5 dB Improvement ≤4 dB Worsening ≤4 dB Improvement ≥5 dB Worsening 58 Conclusion: Early Clinical Proof of Concept Established and Supports Initiation of Expansion Study

• Improvement: 3/3 patients in C3 and 2/6 in cohort 4 and cohort 5 exhibited early efficacy signals ▪ Consistency of results demonstrated across different microperimetry efficacy analyses and timepoints

• Maintenance: 93% of treated/untreated eyes maintained visual acuity ▪ No significant declines in ellipsoid zone or microperimetry sensitivity observed

• Safety: NSR-RPGR was observed to be generally well tolerated ▪ Steroids may rescue efficacy dampened by inflammation

59 Next Steps: XIRIUS Phase 1/2 Expansion Study to Initiate Q4 2018

• Expansion study to enroll from 7 Expansion Study sites in US/UK ▪ Oxford ▪ Oregon Follow- Target Dose, (n=30) ▪ Manchester ▪ UPenn up Visits Randomization ▪ Southampton ▪ Univ. of Florida 2:1 allocation ratio ▪ Miami Follow- Low-Dose Control, (n=15) up Visits

• Includes pediatric patients • Preliminary data expected mid 2019

Note: FDA draft guidance “Human Gene Therapy for Retinal Disorders” published 7/12/18. * 6 dose cohorts of 3 patients each 60 Q&A: NSR-RPGR Moderated by: Senthil Sundaram Email questions to Chief Financial Officer, Nightstar Therapeutics [email protected]

8:50 AM – 9:05 AM NSR-REP1 for the Tuyen Ong, M.D, MRCOphth, MBA Treatment of EVP , Chief Development Officer, Choroideremia Nightstar Therapeutics

9:05 AM to 9:15 AM Choroideremia: Rare, Degenerative Disease That Leads to Blindness With No Treatment Options

Normal Retina • X-linked inherited mutation in CHM gene which encodes Rab escort protein-1 (REP1) ▪ Orphan disease - 1:50,000 Macula Optic Disc • REP1 protein plays key role in intracellular protein trafficking and elimination of waste products from retina • Absence of functional REP1 leads to death of RPE cells and degeneration of photoreceptors • Disease progression Choroideremia Retina ▪ Early - Night blindness Age 57; 76 ETDRS BCVA ▪ Mid - Peripheral vision loss ▪ Late - Central vision loss ▪ Can impact patient’s work, school and social interactions

Source: MacDonald et al., Choroideremia, Feb 2003.. Retina is pale due to extensive 63 loss of retinal cells/choroid Choroideremia Disease Progression Over Time

Images courtesy of the Choroideremia Research Foundation. 64 Maintenance of Visual Acuity is the Primary Clinical Goal Corresponding • Visual acuity declines in untreated Nightstar Trials N C K Z O choroideremia patients NIGHT Natural History Study • Primary clinical goal is maintenance R H S D K of visual acuity REGENERATE D O V H R ▪ Meaningful for patients Phase 2 Trial • Regulatory strategy for approval C R Z H S ▪ Hyper-responder: STAR ONHRC Gain of ≥15 letters (3 lines) Phase 3 Trial DKSNV Z S O K N ⬧ FDA accepted endpoint C K D N R S R Z K D H Z O V C ⬧ 2-Year data from Phase 1/2 clinical trial NVDOK VHCNO SVHCZ Z V D O K

65 Over 90% of NSR-REP1 Treated Patients Maintained Visual Acuity Untreated patients have >2.5x risk of visual acuity loss based on two year timepoint

One Year Follow-up Two Year Follow-up NSR-REP1: High Dose Untreated 22%

13%

letters loss letters -

8% 8% % Eyes with ≥5 with Eyes %

2/26 40/308 2/26 68/308 NSR-REP1 ISTs: High Dose NIGHT NSR-REP1 ISTs: High Dose NIGHT 1 Year (n=26) 12 Month (n=308) 2 Year (n=26) 20 Month (n=308) Note: ISTs = Investigator Sponsored Trials. Maintenance of VA = loss of less than 5 ETDRS letters. Charts based on retrospective analyses of data from NSR-REP1 ISTs. 66 21% Hyper-response (15-letter Gainer) Observed in Treated Patients in Phase 1/2 Trials

Hyper-response Unlikely in Untreated Patients 21% NSR-REP1

Untreated

letter gain letter

- % Eyes with ≥15 with Eyes %

1% n=4 n=3 Phase 1/2 at 1 Year NIGHT at 1 Year (n=19) (n=206)

Note: Retrospective analysis. Baseline VA 34-73 subgroup is optimal for assessing VA gain (avoids ceiling effect). 67 Safety Profile: NSR-REP1 Well-Tolerated

• Current data from 32 treated patients in the completed investigator sponsored trials (ISTs) indicate NSR-REP1 was well tolerated • Safety profile consistent with vitrectomy ▪ Adverse events of varying severity and duration generally resolve within one week ⬧ retinal changes, intraocular inflammation and visual disturbances • 2 Treatment-related Serious Adverse Events ▪ 1 Possibly drug-related: intraocular inflammation ▪ 1 Procedure-related: gas bubble in surgical tubing

68 NSR-REP1 for the Robert MacLaren, Treatment of MBChB, D.Phil., FACS Professor of Ophthalmology, University of Oxford Choroideremia Scientific Founder, Nightstar Therapeutics 9:15 AM – 9:20 AM Oxford Experience Sustained Visual Acuity Improvements w/ NSR-REP1 Treatment Over Time Group plots of median changes Individual plots of visual acuity changes

Treated group of eyes blue compared to All 12 eyes treated per protocol maintained or untreated green Wilcoxon signed rank improved visual acuity (green) whereas 8 of 12 test, n=12 untreated eyes have lost vision (red) 70 Source: Xue et al., Nature Medicine (in press, Oct 2018). Durable Fixation Shift Measured Using Microperimetry

Source: Xue et al., Nature Medicine (in press, Oct 2018). Asterisk indicates patients who subsequently had cataract 71 surgery L1, L3, H1, H6 and H7 (n=5) have mild cataracts developing. Hybrid CMC Model in Julian Hanak Gene Therapy Senior Vice President, Global Head of CMC, Nightstar Therapeutics 9:20 AM to 9:30 AM Hybrid CMC Model: In-house Product Development & Quality Assurance With Third-Party Facilities & Laboratories

GMP Materials & Manufacturing GMP Testing by Leading CMO & CRO Central Labs from Leading CMOs Reduces process establishment time & costs Tech De-risks CMO/CRO success transfer Oversight

Nightstar Proprietary Process Nightstar Quality Assurance & Analytical Development and Regulatory Affairs

73 Ocular Gene Therapy 70 to 700x Lower Product Requirement for AAV Vector Compared to Systemic Gene Therapy

100 Subretinal Gene Therapy Doses 100 Systemic Gene Therapy Doses 1 x 1013 to 1 x 1014 Vector Particles Up to 7 x 1015 Vector Particles1

80L 5,600L Maximum Minimum process Process volume2 volume2

Sources: (1)Calculated from Duan, Molecular Therapy, July 2018. (2) Assuming similar productivity is maintained upon process scale-up 74 Production of Phase 3 Drug Using Commercial-Scale Process

TFF1

Thaw Cells & Transfection Transfect Cells Benzonase Virus Harvest Lysate Freeze Clarification & Concentration Expand Cell in Media with Plasmid (Cell Lysis) (Hold step) by UF/DF Disposable DNA & Benzonase Cell Factories Treatment

TFF3 TFF2

Fill & Finish of Drug Drug Concentration and Final Eluate Chromatography Ultra- Product Substance Formulation Secondary centrifugation Purification Primary Purification Note: For illustrative purposes only. Actual manufacturing process and steps may differ and be modified from time-to-time. Abbreviations: TFF - tangential flow ultrafiltration; 75 UF – ultrafiltration; DF – diafiltration. GMP Process Improvements: Cell Banks

• Proprietary GMP Master and Working Cell Banks ▪ HEK293 cells ▪ Fully traceable to ATCC confirming cell line origin ▪ Commercial license from originator ▪ Cell banks created under conditions of GMP ▪ Well-tested Cell Banks ▪ Well-characterized ▪ Tested for absence of adventitious agents ▪ Comparability demonstrated to NCH cell bank

Abbreviations: GMP – Good Manufacturing Practice; ATCC – American Type Culture Collection; HEK – Human embryonic kidney 76 GMP Process Improvements: HEK293 Triple Transfection

▪ Two-fold scale-up in plastic disposable cultureware ▪ Redesigned plasmids to remove Ampicillin resistance gene ▪ GMP plasmids used for NSR-REP1 ▪ Removal of chloroquine treatment step

Transfection Media Transfect Cells ▪ Harvest point optimized for AAV yield and quality with Plasmid DNA

77 GMP Process Improvements: Upstream Freeze/Hold

• Introduction of upstream hold step to separate from downstream process ▪ Improves GMP logistics and plant flexibility ▪ Allows in-process testing & de-risks downstream purification ▪ Facilitates campaign stockpiling (multiple batches/overlapping batches)

Freeze (Hold step)

78 GMP Process Improvements: Ultrafiltration & Diafiltration

• Cross-flow filtration TFF1 ▪ Tangential flow ultrafiltration (TFF) ▪ Replaces unscalable dialysis ⬧ Allows filtration and buffer exchange in single step ▪ Fast, measurable and controllable ▪ GMP compliant

79 GMP Process Improvements: Chromatography

• Replacement of Chromatography matrix ▪ Removal of porcine heparin ▪ Replacement with AAV affinity chromatography ▪ Removal of low salt step

Chromatography Secondary Purification

80 GMP Process Improvements: Fill/Finish

Final product presentation ▪ Double-redundant sterile filtration ▪ Commercial pharmaceutical presentation ⬧ Glass vials with tamper-evident flip-tops

Fill & Finish of Drug Product

81 GMP Process Improvements: Quality Control

Quality control and control strategy ▪ Increased in-process tests ▪ Assay validation ▪ Development of new assay ⬧ Specific potency assay ⬧ Residuals analysis ⬧ Total particle assay ▪ Quantitative specifications agreed with regulators

82 Collaboration with Cell & Gene Therapy Catapult UK to Manufacture NSR-RPGR

• Secured £1.5 million in funding from Innovate UK, UK’s innovation agency

• Industrial research project for commercial manufacture of NSR-RPGR

• Work initiated in April 2018

83 NSR-REP1 Phase 3 Material Manufactured at Commercial Scale Using GMP Process

• Hybrid manufacturing model de-risks CMO supply and balances costs • Experienced Nightstar CMC team • GMP and process improvements introduced and implemented for Phase 3 • NSR-REP1 analytical testing developed and fit for Phase 3 ▪ Characterization and validation of current process underway in anticipation of potential BLA and commercialization

84 NSR-ABCA4 for the Robert MacLaren, Treatment of Stargardt MBChB, D.Phil., FACS Professor of Ophthalmology, University of Oxford Disease Scientific Founder, Nightstar Therapeutics 9:30 AM – 9:35 AM Stargardt Disease: Progressive Photoreceptor Degeneration that Leads to Blindness; No Treatment Options

• Most common form of inherited juvenile Stargardt Disease macular dystrophy Fundus of Patient with Mid-Stage Disease • Orphan Disease @ 1:10,000 ▪ 65,000 Stargardt patients in U.S./EU5 • ABCA4 transmembrane protein plays key role in clearing away toxic byproducts from visual cycle ▪ Absence of functional ABCA4 leads to degeneration of photoreceptors • Can lead to blindness in patients in twenties ▪ Develops during childhood and adolescence

Source: National Eye Institute website. 86 ABCA4 Clears Toxic Metabolites from Photoreceptor Outer segment Discs

• Outer segment discs contain light sensing protein (rhodopsin)

OS • ABCA4 is a transmembrane protein in the OS discs CC ▪ controls export of certain toxic Rhodopsin ABCA4 visual cycle byproducts IS • ABCA4 mutations associated with All-trans-retinal Phosphatidylethanolamine Retinoid ADP buildup of toxins in N-retinylidene- phosphatidylethanolamine Cycle ATP photoreceptors and RPE ▪ all-trans-retinal ▪ bisretinoids ▪ lipofuscin

Source: Sears et al., TVST Sep 2017. 87 Double to Single Stranded DNA

5’ + 3’

3’ - 5’

88 Single to Double Stranded DNA

NUCLEUS

DNA transcription DNA transcription 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’

89 The Big Gene Question

5’ + 3’

3’ - 5’

5’

90 The Big Gene Question

NUCLEUS

5’ 3’ 3’ 5’

91 NSR-ABCA4: Dual-vector Approach Utilizing Optimized Overlap Region

NUCLEUS

DNA transcription

5’ 3’

3’ 5’

92 NSR-ABCA4: Dual Vector Restores ABCA4 to Retina in Preclinical Model

Wildtype (WT), ABCA4 knockout (KO) show positive and negative controls. ABCA4 marks the outer segments whereas Hcn1 labels the inner segments. Upstream KO (single vector at 5’ end), Downstream KO (single AAV vector from 3’ end) show no ABCA4 production as expected, but Dual KO (combined AAV vectors) leads to robust ABCA4 protein expression (E and F). 93 NSR-ABCA4: Dual Vector Reduces Autofluorescence in Pre-Clinical Model

6 months post-injection 2.0 *

s h

Treatment injected eye t

n 1.5

n e

200µm c

s e

r 1.0

u l

Sham injected eye f

n 0.5

o F 0.0 Treatment Sham

Increased autofluorescence is an early feature of Stargardt disease. Dual vector treated mice show a reduction in retinal autofluorescence compared with saline injected controls (n=12, paired t-test, p<0.05). Mice were treated in early adult life (<3 months) and retinal autofluorescence imaging (Heidelberg Spectralis) was performed at 3 and 6 months. 94 Q&A Moderated by: Senthil Sundaram Email questions to Chief Financial Officer, Nightstar Therapeutics [email protected]

9:35 AM – 9:45 AM Closing Remarks David Fellows Chief Executive Officer, Nightstar Therapeutics 9:45 AM – 10:00 AM Key Completed and Upcoming Milestones

NSR-REP1 NSR-RPGR ✓Sept. 2018: Preliminary proof-of- ✓March 2018: Initiation of Phase 3 STAR concept data from Dose Escalation Trial Study at EURETINA 2018 2018 ✓April 2018: Two-year data for ISTs • Q4 2018: Initiation of Expansion Study ✓June 2018: RMAT Designation

• Q2 2019: Six-month follow-up data from Dose Escalation Study • H1 2019: Full enrollment for Phase 3 • Mid 2019: Preliminary data from 2019 STAR Trial Expansion Study • Q4 2019: One-year follow-up data from Dose Escalation Study

• 2020: One-year follow-up data from • 2020: One-year follow-up data from 2020 Phase 3 STAR Trial Expansion Study Note: Green check mark (✓ ) denotes completed milestones, all other milestones are anticipated future milestones. 97 4 Reasons To Believe

1. CHM: Farthest along with Phase 3 ongoing

2. XLRP: Phase 1/2 Proof-of-Concept

3. ABCA4: Next pipeline program from Oxford

4. Multiple potential pipeline catalysts in 2018, 2019 and 2020