Maintain & Restore Vision

Nightstar Therapeutics R&D Day

September 24, 2018

Email your questions to: Investors@nightstartx.com

* 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.

2

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 toidentify forward-looking statements, although not all forward-looking statements contain these identifying words. All statements contained in thispresentation other than statements of historical facts are forward-looking statements, including, without limitation: our planned and ongoing clinical trials forNSR-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 andtimelines relating to the dose escalation study in the XIRIUS trial and the planned expansion study in the XIRIUS trial; the potential utility of prior preclinicaland clinical data and the data and endpoints presented herein in predicting future clinical results for our product candidates and any results of assessments tobe 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; thecontinued clinical development of our pipeline; the timelines associated with our research and development programs including the timing of patientenrollment and the release of data from ongoing clinical trials and studies; the prevalence of patient populations for our targeted indications; and statementsabout 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 anumber of involve substantial known and unknown risks, uncertainties and other factors that may cause our actual results, levels of activity, performance orachievements to be materially different from the information expressed or implied by these forward-looking statements, including those related to the timingand costs involved in commercializing any product candidate that receives regulatory approval; the initiation, timing and conduct of clinical trials; theavailability of data from clinical trials and expectations for regulatory submissions and approvals; whether interim results of a clinical trial will be predictive ofthe 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 andgeneral development progress; the availability or commercial potential of the our product candidates; the sufficiency of our cash resources; and other risksand 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 subsequentreports that we file with the U.S. Securities and Exchange Commission. We may not actually achieve the plans, intentions, estimates or expectations disclosedin our forward-looking statements, and you should not place undue reliance on our forward-looking statements. Actual results or events could differmaterially from the plans, intentions, estimates and expectations disclosed in the forward-looking statements we make. We anticipate that subsequent eventsand developments will cause our views to change. We are under no duty to update any of these forward-looking statements after the date of thispresentation to conform these statements to actual results or revised expectations, except as required by law. You should, therefore, not rely on theseforward-looking statements as representing our views as of any date subsequent to the date of this presentation. Any reference to our website address in thispresentation is intended to be an inactive textual reference only and not an active hyperlink.

Disclaimers

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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) –Background and Efficacy Endpoints

Byron L. Lam, M.D., Greene Professor of Ophthalmology, Bascom Palmer Eye Institute, University of Miami School of Medicine

8:20 – 8:50 AM XIRIUS Phase 1/2 Trial: Dose Escalation Study Preliminary Results

Robert MacLaren, MBChB, D.Phil., FACS, Professor of 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 Choroideremia

Tuyen Ong, M.D., MRCOphth, MBA, Chief Development Officer, Nightstar TherapeuticsRobert 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 StargardtDisease

Robert MacLaren, MBChB, D.Phil., FACS, Professor of 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

Our Mission: Maintain and Restore Vision in Blinding Inherited Retinal Diseases

David Fellows

Chief Executive Officer,Nightstar Therapeutics

8:00 AM to 8:10 AM

5

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

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We retain worldwide commercial rights to all of our product candidates

Advanced Pipeline of Novel Gene Therapy Candidates Targeting Inherited Retinal Diseases

Program IndicationPrevalence(US/EU5)1

Stage of Development Next Milestone

NSR-REP1 Choroideremia13K

(1:50K)Phase 3

Complete Phase 3 Enrollment: 1H 2019

NSR-RPGR X-linked Retinitis Pigmentosa17K

(1:40K)Phase 1/2

Initiation of Expansion Cohort: 4Q 2018

NSR-ABCA4 Stargardt Disease65K

(1:10K)Preclinical

NSR-BEST1 Best Disease10K

(1:67K)Preclinical

1 Management estimates based on published epidemiology.

X-Linked Retinitis Pigmentosa -Background and Efficacy Endpoints

Byron L. Lam, M.D.

Greene Professor of Ophthalmology, Bascom Palmer Eye InstituteUniversity of Miami School of Medicine

8:10 AM to 8:20 AM

8

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

▪ Mostly males but some females affected

• 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; (4) Iftikhar et al., Ophthal Retina, May 2018. (Image) Hamel, Orphanet Journal of Rare Diseases, October 2006

XLRP PatientPeripheral “bone spicules” & retinal atrophy

9

Pathology of Disease:RPGR Mutations Can Lead to Photoreceptor Death

Source: Birch, Retinal Physician, April 2018.

• RPGR localized in cilium tract

▪ Connective body between inner and outer segment of photoreceptors

• 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 transport across cilium

BasalBodies

10

Preservation of Photoreceptors: Early Versus Late Effects

Potential Late Effect Retinal Structure: Ellipsoid Zone

Potential Early EffectRetinal Sensitivity: Microperimetry

Treatment area Microperimetry grid

Light Sensitive Retina

Outer Nuclear Layer

Photoreceptor Cells

Inner and Outer Segments

Retinal Pigment Epithelium (RPE)

Bruch’s Membrane

Choroid

Sources: (1) Brightfocus (2) Iftikhar et al., Ophthal Retina, 2018 (3) adapted from Birch et al., JAMA Ophthalmology, Sept. 2013.

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Potential Long-term Endpoint for XLRPEZ Width/Area: Structural Measurements of Degenerative Photoreceptor Loss

• 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” published 7/12/18

Ellipsoid Zone (EZ)RPE inner borderBM / choroid

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Microperimetry (MP): Functional Assessment of Retinal Sensitivity & Potential Early Endpoint for XLRP

Video source: (Left) MAIA Microperimeter General Presentation Video; (Right) Retinal sensitivity map shown is an example of an image from an XLRP patient

Example XLRP Patient Retinal Sensitivity Map

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Microperimetry: Functional Measurement of Macula Retinal SensitivityBetter Marker of Visual Function than Visual Acuity1

Scotoma(s)

Island(s) of Vision

• Retinal sensitivity heatmap• 68 stimulus points• Increased accuracy & sensitivity

• Areas of retina unable to perceive any stimuli

Detailed Retinal Imaging

Least sensitivity

Most sensitivity

XLRP Patient Image

MAIA Microperimetry• Realtime imaging of entire macula• Automated eye tracking• Stimulation of same retinal loci

across tests/visits

Source: (1) Iftikhar et al., Ophthal Retina, 2018.

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Microperimetry: High Sensitivity and Correlated with Vision-related Quality of Life Measurements

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

“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

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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 productSource: Iftikhar et al., Ophthal Retina, 2018.

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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 productSource: Iftikhar et al., Ophthal Retina, 2018.

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Increase in Number of Scotomas Indicative of 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 productSource: Iftikhar et al., Ophthal Retina, 2018.

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Expectation in Untreated Retinitis Pigmentosa Patients: Microperimetry Retinal Sensitivity Decreases Over Time

Source: Iftikhar et al., Ophthal Retina, 2018. Data from Nidek MP-1 using 68 loci pattern.

-0.4 dB

-0.6 dB

-1.3 dB

-1.1 dB

68 loci Central 16 Edge of scotoma Seeing retina

Mean Yearly Decline in Retinal Sensitivity (dB)

XIRIUS Phase 1/2 Trial: Dose Escalation Study Preliminary Results

Robert MacLaren, MBChB, D.Phil., FACS

Professor of Ophthalmology, University of OxfordScientific Founder, Nightstar Therapeutics

8:20 AM – 8:50 AM

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RPGR is Alternatively Spliced

…GGGAGGAGAG15Exons 1-145’ 16 Exons 17-19 3’

GUGAGU AG

Intron 15 donor acceptor

RPGR primary RNA transcript

U1/6 sRNA NUCLEUS

CYTOPLASM

Ubiquitous RPGR mRNA

nuclear membrane nuclear membrane

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..

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RPGR is Alternatively Spliced

…GGGAGGA15Exons 1-145’

GUGAGU AG

Intron 15 donor acceptor

RPGR primary RNA transcript

U1/6 sRNA NUCLEUS

CYTOPLASM

Photoreceptor specific RPGR mRNA - RPGRORF15

16 Exons 17-19 3’GAG

GUGAGU

AG

3’

15Exons 1-145’ 16 Exons 17-19 3’

Ubiquitous RPGR mRNA

nuclear membrane nuclear membrane

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.

22

RPGR Gene Therapy: Problem

…GGCAG15Exons 1-145’

GUGAGU AGAAV8.RPGR mRNA

U1/6 sRNA NUCLEUS

CYTOPLASM

Truncated RPGR mRNA – potentially toxic

3’GA

nuclear membrane nuclear membrane

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

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.

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RPGR Gene Therapy: Solution

GUGUCCAAV8.RPGR mRNA

U1/6 sRNA NUCLEUS

CYTOPLASM

Correct full-length RPGRORF15 mRNA from AAV8 vector

…GGCAG15Exons 1-145’ 3’GA

CodonsAGU=SerineUCC=Serine

nuclear membrane nuclear membrane

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.

24Source: 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.

Codon-optimized NSR-RPGR Yields Correct Full-Length Protein

Dominik Fischer in Oxford

Western blot – RPGR bands corresponding to the full length protein

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ORF15 Region is Functional

RPGRORF15 translated into protein

…GGCAG15Exons 1-145’ 3’GA

COOHNH2 Glu

Gly

Glu

Gly

Glu

Gly

GAA = Glu GAG = GluGGA = GlyGGG = Gly

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.

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RPGR Glutamylation with TTLL5

Glu

Gly

Glu

Gly

Glu

Gly

RPGRORF15 post-translational modification of protein

TTLL5

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. Abbreviation: TTLL5 - tubulin tyrosine ligase like-5

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Glutamylation Moves RPGR

Glu

Gly

Glu

Gly

Glu

Gly

RPGRORF15 post-translational glutamylation

tubulin in photoreceptor cilium

outer segmentinner segment

28

Effect of RPGR ORF15 Deletion

Gly

Glu

Gly

RPGR with ORF15 deletion has reduced glutamylation

TTLL5

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. Abbreviation: TTLL5 - tubulin tyrosine ligase like-5

29

Deleted RPGR is Defective

Gly

Glu

Gly

RPGRORF15 with reduced glutamylation due to deletion

tubulin in photoreceptor cilium

outer segmentinner segment

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.

30

Deleted RPGR is Defective

Glu

Gly

Gly

RPGRORF15 with reduced glutamylation due to deletion

tubulin in photoreceptor cilium

outer segmentinner segment

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 underlies pathology of TTLL5 mutations. Proc Natl Acad Sci 2016.

31

Effects of ORF15 Deletion

Source: Sun et al. Loss of RPGR glutamylation underlies pathology of TTLL5 mutations. Proc Natl Acad Sci 2016.

32

Codon Optimized NSR-RPGR In Vitro

Full length RPGR protein with no

splice variants in codon optimised

RPGR construct (white arrow)

Full length and fully glutamylated

ORF15 seen with GT335 immuno-

staining in codon optimised RPGR

Dr. Cristina Martinez

Nuffield Laboratory

of Ophthalmology,

University of Oxford

CORRECT SPLICING CORRECT GLUTAMYLATION

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.

33

Preservation of Photoreceptors: Early Versus Late Endpoints

Retinal Structure: Late Effect

Retinal sensitivity: Potential Early Effect

Treatment area Microperimetry grid

Light Sensitive Retina1

Outer Nuclear Layer

Photoreceptor Cells

Inner and Outer Segments

Retinal Pigment Epithelium (RPE)

Bruch’s Membrane

Choroid

Sources: (1) Brightfocus (2) Birch et al., JAMA Ophthalmology, Sept. 2013. Abbreviation: EZ – ellipsoid zone

• Utility as LT endpoint2

▪ Mean annual decrease in EZ width of 248 μm (7%)

34

XIRIUS: Phase 1/2 Clinical Trial Design Includes Two Studies

EligibilityGenetically confirmed diagnosis of XLRP

Males 18 years and older Males 10 years and older

Primary Endpoint

Safety: Incidence of dose limiting toxicities & treatment emergent adverse events

Selected Secondary Endpoints

• Maintenance of vision (BCVA)• Changes in microperimetry • Changes in SD-OCT Ellipsoid Zone

Upcoming Milestones

Q2 2019 – Six-month follow-up data expectedQ4 2019 – One-year follow-up data expected

Q4 2018 – Initiation of expansion study expectedMid 2019 – Preliminary data expected

Data Monitoring Committee

Treatment(n=18 total*)

Follow-up Visits

Dose escalation

Randomization2:1 allocation

ratio

Follow-up Visits

Follow-up Visits

Low-Dose Control, (n=15)

Target Dose, (n=30)

Dose Escalation Study Expansion Study

Note: FDA draft guidance “Human Gene Therapy for Retinal Disorders” published 7/12/18.* 6 dose cohorts of 3 patients each

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Baseline Demographics: Generally Comparable Between Treated / Untreated Eyes

Cohort Dose(gp)

# of Pts

Mean Age

Baseline Mean Retinal Sensitivity (dB)

Baseline Mean BCVA (Letters)

Baseline Mean EZ Width (microns)

Treated Eye

Untreated Eye

Treated Eye

Untreated Eye

Treated Eye

Untreated 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.

• 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

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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

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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

38

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

BASELINE MONTH 1

MONTH 3 MONTH 6

0.5 dB 0.7 dB 3.4 dB 0.5 dB

0.5 dB6.6 dB 0.5 dB6.9 dB

Treated Untreated

Treated Untreated Treated Untreated

Treated Untreated

Note: microperimetry images and data from responder in cohort 4. Baseline image shown reflects final image after triplicate testing.

39

BASELINE

0.5 dB 0.7 dBTreated Untreated

Case Example: Durable Improvements In Both Area & Mean Sensitivity

Note: microperimetry images and data from responder in cohort 4. Baseline image shown reflects final image after triplicate testing.

40

MONTH 1

3.4 dB 0.5 dBTreated UntreatedNote: microperimetry images and data from responder in cohort 4.

Case Example: Durable Improvements In Both Area & Mean Sensitivity

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MONTH 3

0.5 dB6.6 dB Treated UntreatedNote: microperimetry images and data from responder in cohort 4.

Case Example: Durable Improvements In Both Area & Mean Sensitivity

42

MONTH 6

0.5 dB6.9 dB Treated UntreatedNote: microperimetry images and data from responder in cohort 4.

Case Example: Durable Improvements In Both Area & Mean Sensitivity

43

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

44

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

45

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

0.10.2

2.4

0.10.60.2

-0.8

-0.1 -0.1

1.2

-2

-1

0

1

2

3

4

5

Cohort 1 Cohort 2 Cohort 3 Cohort 4 Cohort 5

Change in Mean Sensitivity (dB) at Month 1 Treated Untreated

2dB Threshold

n=3 n=2 n=3 n=3 n=3Note: 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). One patient in cohort 2 was excluded from the analysis because triplicate testing was not performed at baseline.

3 responders

1 responder

1 responder

46

Central 16 Sensitivity:Greater Efficacy Signal Seen (~ 6 dB in Cohort 3)

0.5

-0.1

6.1

1.2

-0.6

0.4

-0.8 -1.0-0.2

1.4

-5

-3

-1

1

3

5

7

9

Cohort 1 Cohort 2 Cohort 3 Cohort 4 Cohort 5

Change in Mean Sensitivity of Central 16 Loci (dB) at Month 1Treated Untreated

n=3 n=2 n=3 n=3 n=3Note: 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). One patient in cohort 2 was excluded from the analysis because triplicate testing was not performed at baseline.

47

Number of Improved Loci:Responders in Cohorts 3-5 (≥ 5 dB Improvement at 10% of Loci)

0

1

3

1 1

0 0 0 0

1

Cohort 1 Cohort 2 Cohort 3 Cohort 4 Cohort 5

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

Treated Untreated

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.

48

Inflammation Dampened Efficacy in C4-C5 Treated Eyes…

0

1

3

1 1

Cohort 1 Cohort 2 Cohort 3 Cohort 4 Cohort 5

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

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.

Inflammation May Have Dampened Efficacy

Lower Dose & More Severe Patients

49

Case Example: Steroids Rescued Efficacy Dampened by Inflammation

0

2

4

6

8

Baseline Month 1 Week 5 Month 3 Month 6

Me

an R

etin

al S

en

siti

vity

(d

B)

Months Following NSR-RPGR Administration

Treated Untreated

Note: microperimetry data from responder in cohort 4. Microperimetry data for untreated eye at week five not available.

Interim visit due to inflammation

Sensitivity Map for Treated Eye

Treatment with oral steroids

XIRIUS Trial Dose Escalation Study: Review of Cohort 3 Data

Robert MacLaren, MBChB, D.Phil., FACS

Professor of Ophthalmology, University of OxfordScientific Founder, Nightstar Therapeutics

9:15 AM – 9:20 AM

51

Consistency of Results Observed Across Different Efficacy Analyses in Cohort 3

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).

2.4

-0.1

Change in Mean Sensitivity (dB)

100%

0%

Patients with 5 dB Improvement at 10% Loci

6.1

-1.0

Change in Mean Sensitivity of Central 16 Loci (dB)

Treated Untreated

52

Overall Mean Sensitivity in Treated Eyes Improved Compared to Untreated to Untreated Eyes in Cohort 3

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).

2.4 2.4 2.3

0.0

-0.1-0.3 -0.5

-3

-2

-1

0

1

2

3

4

5

Baseline Month 1 Month 3 Month 6

Ch

ange

in M

ean

Ret

inal

Se

nsi

tivi

ty (

dB

)

Months Following NSR-RPGR Administration

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

53

Central 16 Mean Sensitivity in Treated Eyes Improved by ≥6 dB Compared to Untreated Eyes in Cohort 3

6.15.6

5.1

-1.0 -1.1 -1.2

-4

-2

0

2

4

6

8

10

Baseline Month 1 Month 3 Month 6

Ce

ntr

al 1

6 L

oci

Ch

ange

fro

m B

ase

line

in M

ean

Se

nsi

tivi

ty

Months Following NSR-RPGR Administration

Treated Eye Untreated Eye

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).

(n=3) (n=3)

54

100% 100% 100%

0% 0% 0%0%

20%

40%

60%

80%

100%

Baseline Month 1 Month 3 Month 6

% P

atie

nts

wit

h M

inim

um

of

5 d

B

Ch

ange

in 1

0%

of

Loci

Months Following NSR-RPGR Administration

Treated Eye Untreated Eye

≥5 dB Improvement in Retinal Sensitivity in More than 10% of Loci in Treated Eyes But Not Untreated Eyes in Cohort 3

Note: The analysis is based on difference in sensitivities between baseline and six month follow-up.

(n=3) (n=3)

55

30

27 26

20

1513

1013

97

42 1 1

≥1 dB ≥2 dB ≥3 dB ≥4 dB ≥5 dB ≥6 dB ≥7 dB

Month 1

Treated Untreated

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

27

2422

19

16

1312

86

53

1 10

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

Month 6

NSR-RPGR Treated Eyes: Increases in Retinal Sensitivity Generally Sustained Across Various Thresholds

56

11

0 0 0 0 0

1211

8

7

4

3

1

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

Month 1

Treated Untreated

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

2

1 1 10 0 0

13

12

98

6

5

3

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

Month 6

Untreated Eyes: More Loci With Decreases in Retinal Sensitivity

57

All Treated Eyes in Cohort 3 Showed Improvement in Retinal Sensitivity

Treated Eyes Untreated Eyes

C3-1 C3-2 C3-3M

on

th S

ix

M

on

th O

ne

Bas

elin

eNote: Baseline images shown reflect triplicate testing.

C3-1 C3-2 C3-3

58

NSR-RPGR Treated Eyes Showed Early Improvements at Month 1 with Sustained Effects to Month 6

No Change≤4 dB Worsening≥5 dB Worsening

Legend: ≥5 dB Improvement≤4 dB Improvement

Treated Eyes Untreated Eyes

Mo

nth

Six

M

on

th O

ne

C3-1 C3-2 C3-3 C3-1 C3-2 C3-3

59

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

60

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

Randomization2:1 allocation

ratio

Follow-up Visits

Follow-up Visits

Low-Dose Control, (n=15)

Target Dose, (n=30)

Expansion Study

Note: FDA draft guidance “Human Gene Therapy for Retinal Disorders” published 7/12/18.* 6 dose cohorts of 3 patients each

• Expansion study to enroll from 7 sites in US/UK

▪ Oxford

▪ Manchester

▪ Southampton

▪ Miami

• Includes pediatric patients

• Preliminary data expected mid 2019

▪ Oregon

▪ UPenn

▪ Univ. of Florida

Q&A: NSR-RPGR

Email questions to investors@nightstartx.com

Moderated by:

Senthil Sundaram

Chief Financial Officer, Nightstar Therapeutics

8:50 AM – 9:05 AM

NSR-REP1 for the Treatment of Choroideremia

Tuyen Ong, M.D, MRCOphth, MBA

EVP , Chief Development Officer, Nightstar Therapeutics

9:05 AM to 9:15 AM

63

Choroideremia: Rare, Degenerative DiseaseThat Leads to Blindness With No Treatment Options

• X-linked inherited mutation in CHM gene which encodes Rab escort protein-1 (REP1)

▪ Orphan disease - 1:50,000

• 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

▪ Early - Night blindness

▪ Mid - Peripheral vision loss

▪ Late - Central vision loss

▪ Can impact patient’s work, school and social interactions

OpticDisc

Macula

Normal Retina

Age 57; 76 ETDRS BCVA

Choroideremia Retina

Retina is pale due to extensive loss of retinal cells/choroid

Source: MacDonald et al., Choroideremia, Feb 2003..

64

Choroideremia Disease Progression Over Time

Images courtesy of the Choroideremia Research Foundation.

65

Maintenance of Visual Acuity is the Primary Clinical Goal

• Visual acuity declines in untreated choroideremia patients

• Primary clinical goal is maintenance of visual acuity

▪ Meaningful for patients

• Regulatory strategy for approval

▪ Hyper-responder: Gain of ≥15 letters (3 lines)

⬧ FDA accepted endpoint

⬧ 2-Year data from Phase 1/2 clinical trial

CN K ZO

R H S D K

D O V H R

C ZR H S

ONHRCDKSNV

Z S O K NCK DN R

S R Z K D

H Z O V C

NVDOKVHCNOSVHCZ

OZ DV K

R H S D K

D O V H R

C ZR H S

ONHRCDKSNV

Z S O K NCK DN R

S R Z K D

H Z O V C

NVDOKVHCNOSVHCZ

OZ DV K

CN K Z

Corresponding Nightstar Trials

NIGHT Natural History Study

REGENERATE Phase 2 Trial

STAR Phase 3 Trial

66

Over 90% of NSR-REP1 Treated Patients Maintained Visual Acuity

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.

8% 8%

13%

22%

NSR-REP1 ISTs: High Dose1 Year (n=26)

NIGHT12 Month (n=308)

NSR-REP1 ISTs: High Dose2 Year (n=26)

NIGHT20 Month (n=308)

NSR-REP1: High Dose Untreated

% E

yes

wit

h ≥

5-l

ette

rs lo

ssUntreated patients have >2.5x risk of visual acuity loss based on two year timepoint

2/26 68/30840/308 2/26

Two Year Follow-upOne Year Follow-up

67

21%

1%

Phase 1/2 at 1 Year(n=19)

NIGHT at 1 Year(n=206)

% E

yes

wit

h ≥

15

-let

ter

gain

21% Hyper-response (15-letter Gainer) Observed in Treated Patients in Phase 1/2 Trials

n=4 n=3

Note: Retrospective analysis. Baseline VA 34-73 subgroup is optimal for assessing VA gain (avoids ceiling effect).

Hyper-response Unlikely in Untreated Patients

NSR-REP1

Untreated

68

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

NSR-REP1 for the Treatment of Choroideremia

Oxford Experience

Robert MacLaren,MBChB, D.Phil., FACS

Professor of Ophthalmology, University of OxfordScientific Founder, Nightstar Therapeutics

9:15 AM – 9:20 AM

70

Sustained Visual Acuity Improvements w/ NSR-REP1 Treatment Over Time

All 12 eyes treated per protocol maintained or improved visual acuity (green) whereas 8 of 12

untreated eyes have lost vision (red)

Treated group of eyes blue compared to untreated green Wilcoxon signed rank

test, n=12

Group plots of median changes Individual plots of visual acuity changes

Source: Xue et al., Nature Medicine (in press, Oct 2018).

71

Durable Fixation Shift Measured Using Microperimetry

Source: Xue et al., Nature Medicine (in press, Oct 2018). Asterisk indicates patients who subsequently had cataract surgery L1, L3, H1, H6 and H7 (n=5) have mild cataracts developing.

Hybrid CMC Model in Gene Therapy

Julian Hanak

Senior Vice President, Global Head of CMC, Nightstar Therapeutics

9:20 AM to 9:30 AM

73

Hybrid CMC Model: In-house Product Development & Quality Assurance With Third-Party Facilities & Laboratories

Nightstar Proprietary Process & Analytical Development

GMP Materials & Manufacturing from Leading CMOs

GMP Testing by Leading CMO & CRO Central Labs

Nightstar Quality Assurance and Regulatory Affairs

Reduces process establishment time & costsDe-risks CMO/CRO successTech

transfer Oversight

74

Ocular Gene Therapy 70 to 700x Lower Product Requirement for AAV VectorCompared to Systemic Gene Therapy

100 Subretinal Gene Therapy Doses 1 x 1013 to 1 x 1014 Vector Particles

100 Systemic Gene Therapy DosesUp to 7 x 1015 Vector Particles1

80LMaximum

process volume2

5,600LMinimum

Process volume2

Sources: (1)Calculated from Duan, Molecular Therapy, July 2018. (2) Assuming similar productivity is maintained upon process scale-up

75

Ultra-centrifugation

Primary Purification

Chromatography Secondary

Purification

Drug Substance

Transfect Cellswith Plasmid

DNA

Virus Harvest (Cell Lysis)

& BenzonaseTreatment

Clarification & Concentration by UF/DF

Fill & Finish of Drug Product

LysateBenzonaseThaw Cells &Expand Cell in

DisposableCell Factories

Transfection Media

EluateConcentration and Final Formulation

TFF1

TFF2TFF3

Production of Phase 3 Drug Using Commercial-Scale Process

Freeze(Hold step)

Note: For illustrative purposes only. Actual manufacturing process and steps may differ and be modified from time-to-time. Abbreviations: TFF - tangential flow ultrafiltration;UF – ultrafiltration; DF – diafiltration.

76

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

▪ 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

Cell Banks

77

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

▪ Harvest point optimized for AAV yield and qualityTransfect Cellswith Plasmid DNA

Transfection Media

78

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)

79

GMP Process Improvements: Ultrafiltration & Diafiltration

• Cross-flow filtration

▪ Tangential flow ultrafiltration (TFF)

▪ Replaces unscalable dialysis

⬧ Allows filtration and buffer exchange in single step

▪ Fast, measurable and controllable

▪ GMP compliant

TFF1

80

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

81

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

82

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

83

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

84

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

NSR-ABCA4 for the Treatment of StargardtDisease

Robert MacLaren, MBChB, D.Phil., FACS

Professor of Ophthalmology, University of OxfordScientific Founder, Nightstar Therapeutics

9:30 AM – 9:35 AM

86

Stargardt Disease: Progressive Photoreceptor Degeneration that Leads to Blindness; No Treatment Options

• Most common form of inherited juvenile macular dystrophy

• 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

Fundus of Patient with Mid-Stage Disease

Stargardt Disease

Source: National Eye Institute website.

87

ABCA4 Clears Toxic Metabolites from Photoreceptor Outer segment Discs

Source: Sears et al., TVST Sep 2017.

• Outer segment discs contain light sensing protein (rhodopsin)

• ABCA4 is a transmembrane protein in the OS discs

▪ controls export of certain toxic visual cycle byproducts

• ABCA4 mutations associated with buildup of toxins in photoreceptors and RPE

▪ all-trans-retinal

▪ bisretinoids

▪ lipofuscin

CC

OS

ISRhodopsin ABCA4

All-trans-retinal

Phosphatidylethanolamine

N-retinylidene-phosphatidylethanolamine

Retinoid Cycle

ADP

ATP

88

Double to Single Stranded DNA

5’ 3’

3’ 5’

+

-

89

NUCLEUS

DNA transcription

DNA transcription

Single to Double Stranded DNA

3’ 5’

5’ 3’

3’ 5’

5’ 3’

90

3’ 5’

+

-

5’

5’ 3’

5’

The Big Gene Question

91

NUCLEUS

The Big Gene Question

3’ 5’

5’ 3’

92

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

NUCLEUS

3’ 5’

5’ 3’

DNA transcription

93

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).

94Increased 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.

Treatment Sham0.0

0.5

1.0

1.5

2.0

Fold

change in 7

90nm

auto

fluore

scence b

etw

een 3

& 6

month

s

*

200µm

Treatment injected eye

Sham injected eye

6 months post-injection

NSR-ABCA4: Dual Vector Reduces Autofluorescence in Pre-Clinical Model

Q&A

Email questions to investors@nightstartx.com

Moderated by:

Senthil Sundaram

Chief Financial Officer, Nightstar Therapeutics

9:35 AM – 9:45 AM

Closing Remarks David Fellows

Chief Executive Officer,Nightstar Therapeutics

9:45 AM – 10:00 AM

97

Key Completed and Upcoming Milestones

Note: Green check mark (✓ ) denotes completed milestones, all other milestones are anticipated future milestones.

NSR-REP1 NSR-RPGR

2018

✓March 2018: Initiation of Phase 3 STAR Trial

✓April 2018: Two-year data for ISTs

✓June 2018: RMAT Designation

✓Sept. 2018: Preliminary proof-of-concept data from Dose Escalation Study at EURETINA 2018

• Q4 2018: Initiation of Expansion Study

2019• H1 2019: Full enrollment for Phase 3

STAR Trial

• Q2 2019: Six-month follow-up data from Dose Escalation Study

• Mid 2019: Preliminary data from Expansion Study

• Q4 2019: One-year follow-up data from Dose Escalation Study

2020• 2020: One-year follow-up data from

Phase 3 STAR Trial• 2020: One-year follow-up data from

Expansion Study

98

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