Viral vectors in clinical therapies (3) 7 December 2015

Prof. Józef Dulak, PhD, DSc Department of Medical Biotechnology Faculty of Biochemistry, Biophysics and Biotechnology Room 3.025/3.07 Phone 664-63-75

Email: [email protected]

Drugs/gene therapy delivery to the eye Leber’s congenital amaurosis –

1. Most common cause of congenital blindness in children.

2. LCA2 – one of the forms – caused by in the retinal pigment epithelium-specific 65-kD gene (RPE65).

3. RPE65 is required to keep light-sensing photoreceptor cells – the rodes and cones of the retina – in operating order

4. The RPE65 gene encodes for the isomerohydrolase that isomerizes bleached all-trans-retinal into photosensitive 11-cis-retinal (Jin et al., 2005; Moiseyev et al., 2005). If no 11-cis-retinal is produced due to loss of or impaired RPE65 function, the chromophore rhodopsin cannot be assembled, and the photoreceptors remain insensitive to light stimuli

5. LCA2 is a rare diseases – in USA only 2000 people – but is untreatable and causes blindness early in life

4 LCA gene therapy - AAV 2

Lancet, October 2009 5 – another type of blindness

Choroideremia derives its name from the almost complete loss of the retina, choroid, and retinal pigment epithelium that leads to exposure of the underlying white sclera, which is a unique feature compared with other retinal degenerations.

• an incurable X-linked recessive degenerative disease of the retina and choroid • It has a prevalence of about 1:50 000, with northern Finland having the highest • Loss of night vision begins in the first decade of life and progresses with a gradual loss of peripheral vision and legal blindness by the fi fth decade. • Choroideremia is caused by in the CHM gene, which was one of the first identified by use of positional cloning • Subsequently, prenylation deficiency due to absence of Rab escort protein-1 (REP1) encoded by CHM was identified as the cause of retinal degeneration in choroideremia • Nearly all reported cases of choroideremia so far have been attributed to functionally null mutations • That, combined with the slow rate of degeneration and small size of the CHM protein coding sequence (1.9 kb), make gene therapy with AAV vectors an appealing treatment strategy.

RE MaclLaren et al., Lancet 2014; 383: 1129–37 Gene therapy of choroideremia

• In The Lancet , Robert MacLaren and colleagues present data for six patients treated with subretinal delivery of an AAV vector encoding CHM , the gene disrupted in choroideremia, in a phase 1 trial.

• All patients recovered to baseline visual acuity despite detachment of the fovea as part of the surgical procedure.

• The authors did not notice any obvious detrimental effects resulting from detachment and treatment of the fovea, which is paramount since the ultimate goal of such an intervention is to prevent central visual loss.

• Two of the patients gained significant visual acuity.

HPN Scholl & JA Sahel, Lancet January 16, 2014 Gene therapy trials for various occular diseases

Retinoblastoma – tumor Age-related macular degeneration – AMD – application of PEDF – pigment epithelium derived factor – this is an anti-angiogenic factor (to be discussed during next week lecture)

Leber’s congenital amaurosis Choroidermia

8 Gene therapy restores auditory function in deaf mice

. Hearing loss are caused by environmental or genetic dysfunction of the hair cells – the primary sensory cells of the inner ear; . Hair cells convert mechanical stimuli into electric signals; . Hair cells lack the ability to regenerate – their damage or death is cumulative; . Current treatment: hearing aids or cochlear implants – provide incomplete restoration of function in a limited patient population

scienceblogs.com

www.wku.edu www.drew-fuller.com Gene therapy restores auditory function in deaf mice (2)

1. Restoration of auditory function in mice lacking vesicular glutamate transporter 3 (VGLUT3) - AAV vectors used; however, VGLUT3 mutations are not common in humans 2. Mice with mutations of transmembrane channel-like gene 1 (Tmc1). Mutations in this gene account for 4-8% of genetic deafness in some populations. 40 TMC1 mutations identified that cause deafness in human populations (the mutations in mice are named Beethoven) Tmc1 or its close ortholog, Tmc2, can be used for gene therapy - adenoviral vectors with Tmc1 or Tmc2 cDNA - AAV 2/1 with Tmc1 or Tmc2

(Tmc1 and Tmc2 appear to be functionally redundant)

Ch. Askew et al., Sci Trans Med. 8 July 2015

Nature, 27th November 2014

Outlook on Haemophilia Haemophilia – basic facts

Liver – the main physiological site of FVIII & FIX synthesis

- Endothelial cells (liver sinusoidal endothelial cells)

Hemophilia A – mutation in FVIII -80% of cases

Hemophilia B – mutation in FIX - 20% of cases Haemophilia – royal families history Inheritance pattern of haemophilia

Patients with mild hemophilia have 5-10% of normal clotting factor levels and they bleed only in case of severe Haemophilia – basic facts

• treatment: even a few times a week with infusions of concentrated factor (20-100+ infusions/year)

• Problems: necessity of repeating, continuous (life-time) injections - risk of infections (HIF, HCV) - cost Treatment of haemophilia

High costs of prophylaxis, the problems associated (viral infections), necessity to inject the factor throuhghout the whole life – justification for the therapy Hemofilia A and B and gene therapy

1. Factor VIII production is not regulated in response to ; 2. Even low levels of the protein can be beneficial 3. The broad therapeutic index of factor VIII minimises the risk of overdoses 4. Delivery of factor VIII into the bloodstream does not require expression of the gene by specific organ;

Size of the coding sequences of factor VIII and factor IX

mRNA - factor VIII - 8,8 kb factor IX - 1,8 kb (or 2.8 kb if long 3’UTR)

17 Clinical gene therapy for

Ex vivo – plasmid gene therapy

Roth DA et al., NEJM 2001; 344: 1735 18 Clinical gene therapy for haemophilia A

Roth DA et al., NEJM 2001; 344: 173519 Effect of ex vivo haemophilia A gene therapy

Roth DA et al., NEJM 2001; 344: 1735 20 AAV8-mediated gene therapy of haemophilia A

B-domain-deleted FVIII cDNA

Clinical trial sponsored by BioMarin has recently begun McIntosh et al., Blood 2013 Ex vivo gene therapy of hemophilia A – lentiviral delivery of factor VIII

- Dogs - HSC transduced with lentiviral vector - Factor VIII released from : a hybrid FVIII molecule fused to the propeptide-D2 domain that traffics FVIII into α-granules - Prevention of severe for at least 2.5 years after gene therapy

Du et al., Nature Commun 2013; J Gould, Nature 27 November 2014 „Christmas” disease

NEJM, December 2011 23 Gene therapy of heamophilia B – AAV2

AAV-mediated factor IX gene transfer to in patients with severe hemophilia B – Manno CS et al., Blood 2003;

Despite strong evidence for gene transfer and expression, circulating levels of F.IX were in all cases less than 2% and most were less than 1% Gene therapy of heamophilia B – AAV2

- Liver delivery – sustained expression, - Muscle injection – no effects

25 Gene therapy of – AAV8

Self-complementary AAV8 vector

26 Gene therapy of haemophilia B – AAV8

1. Self-complementary AAV vector 2. AAV8-pseudotyped 3. Targeting to liver 4. Limitations of immune response (lower prevalence of anti-AAV8 antibodies)

Nathwani et al., NEJM December 2011 27 The higher the dose of a AAV-Factor IX vector, the highest the level of FIX

But higher doses of AAV8 may elicit inflammatory response

Nathawani et al., NEJM, December 2011 28 Current status of gene therapy for hemophilia

K.A. High, Blood 2013 29 Clinical trials for gene therapy in haemophilia – summary

MI Cancio et al., Appl Clin Genetics, 6: 91-101; 2013 Clinical trials for gene therapy in haemophilia – summary

Vectors for hemophilia gene therapy

Hemofilia A

- Plasmid - Retroviral - Adenoviral (not published) - Lentiviral (HSC modification)

Hemofilia B

- AAV vectors - muscle: AAV2 evidence for FIX expression in AAV8 muscle cells up to 10 years after AAV2-FIX administration – but no rise of circulating above 1%)

- Liver - Single strand AAV2 - -self-complementary; AAV8 - Self complementary, AAV8; FIX Padua - Lentiviral (mice)

Nair et al., Advanced Textbook on Gene Therapy, Imperial College, London 2014 Potential complications of AAV-FIX gene therapy for hemophilia

Preventions: limiting the antigen presenting cells transduction

K.A. High, Blood 20123 32 Limitations of AAV gene therapy

High & Anguela, Hum Mol Gene 2015 (Gene) therapy of haemophilia – problems

• About 40% of patients with haemophilia B produce antibodies against AAV – they will be excluded from this type of gene therapy; • Haemophilia A – is more difficult to target by gene therapy (longer gene of factor VIII); • Shortened version of factor VIII is produced; • Still, the strong immune response against AAV8 limits the effectiveness of the therapy - injections of AAV8 cannot be repeated; • The problem may be potentially overcome by ex vivo HSC therapy with lentiviral vector harboring the factor VIII gene; • Potential of gene editing in therapy of haemophilia

• Recombinant-clotting factors – was associated with tens of thousands of haemophiliacs infected with HIV and HCV • About 75% of patients with haemophilia still receive inadequate treatment, particularly in less-developed nations • The fix will not lie in just one solution, but will be contextual and messy

S. Pemberton Is gene therapy for hemophilia necessary?

35 Gene therapy of neurological diseases

About 1,9% of clinical trials are on neurological diseases (around 40 clinical trials)

Some, like Lesh-Nyhan syndrome, although potentially perfect candidate (one dysfunctional gene) are still uncurable

Lentz et al., Neurobiol Dis 2012 Amyotrophic lateral sclerosis

Spinal cord – the atrophy is apparent in ALS Famous AML patients

Lou Gehring Stephen Hawking

1903-1941 1942 – Diagnosed at age of 21 Amyotrophic lateral sclerosis (ALS) (1)

Incidence – 2-3:100 000

Onset at 50-60 years

Sporadic (SALS) – most instances (90-95%) Familial (FALS) – 5-10% - of these 20-25% are mapped to CuZnSOD gene

Degeneration of motor neurons – progressive loss of the ability to move, speak,

Usually fatal within 1-5 years of onset

No effective treatment available Vascular endothelial growth factor (VEGF)

Growth factor that stimulates proliferation and migration of endothelial cells, and increases their survival

40 VEGF-A – a major angiogenic mediator

Matrigel assay

spheroid assay

Receptors on endothelial cells control VEGF

41 Mouse model of hind-limb ischemia

Surgical ligation of the femoral artery)

min

Laser Doppler perfusion measurement

max VEGF165 gene transfer improves blood flow in the rabbit adductor muscle

Blood flow in ischemic adductor muscles 120 p<0.02

100 VEGF

80

60

40 blood flow before ischemia) β−gal -

20 (100% (100% mean mean muscle localflow bloodin adductor 0 Immediately after ligation 14 days after ligation

pSVβ−gal pSG5-VEGF165 transfected transfected

Dulak et al., Eur 2002, 34: 105-110; Józkowicz et al., Int J Artif Organs, 2003: 26: 43161-169 Naked VEGF gene transfer increases the number of microvessels in the ischemic rabbit skeletal muscles

alkaline phosphatase-positive microvessels in muscle transfected with control plasmid

300 2 p<0.01 200

100 Vessels/mm 0 microvessels in muscle β−gal VEGF transfected with transfected transfected VEGF plasmid

44 Dulak et al., Eur Surgery 2002, 34: 105-110; Józkowicz et al., Int J Artif Organs, 2003: 26: 161-169 Neuroprotective functions of VEGF

Oosthuyse et al., Nature Genetics, 28: 131-138, 2001

Both Kdr and Nrp-1 are required

Storkebaum E & Carmeliet P, JCI 2004 Experimental gene therapy for amyotrophic lateral sclerosis

VEGF-expressing lentiviral vector: rabbies G pseudotyped equine infectious anaemia (EIAV) However, this was an experimental approach which so far has not been undertaken in the clinical trials First registered gene therapy drug in Europe.

48 Glybera – first registered AAV vector for human gene therapy

Lipoprotein lipase deficiency – first officialy registered drug – Glybera – accepted by European Commission – 25.10.2012

AAV1 vector with cDNA of lipoprotein lipase (LPL)

A centralised EU marketing authorisation has been obtained under the name Glybera on with the number EU/1/12/791 Lipoprotein lipase deficiency & Glybera

LPLD – type I hyperlipidemia • Ultra-rare disease – 1-2 /106 individuals; • Autosomal recesssive – caused by mutations in LPL gene (chromosome 8p21.3); • LPL – a key in catabolism of triglyceride (TG)-rich lipoproteins; • LPL – secreted from adipocytes and muscle cells; • In LPLD, TG-rich lipoproteins accumulate in the plasma leading to chylomicronemia and severe hypertriglyceridaemia (TG concentration > 20 mmol/l); • This causes episodes of abdominal pain, recurent accute pancreatitis etc.; • Strict diet – dietary fat < 20% of total caloric intake

Glybera • uniQure uses a naturally occurring variant of the LPL gene that has higher enzyme activity than the normal version of the gene that encodes the protein; • The company produces Glybera using its insect cell-based manufacturing proces; • Clinicians administer Glybera in a one-time series of up to 60 intramuscular injections in the legs. The patient is administered spinal anesthesia or deep sedation during the procedurę; • In addition, an immunosuppressive regimen is recommended from three days prior to and for 12 weeks following Glybera administration; To read!

Hildegard Buning

Freely available in PubMed SUMMARY

Gene therapy is effective in a number of monogenic diseases 1.Immunodeficiencies - X-SCID immunodeficiency: retroviral vectors & hematopoietic stem cells - ADA- immunodeficiency - retroviral vectors & hematopoietic stem cells - chronic granulomatous diseases - retroviral vectors & hematopietic stem cells 2. Congential blindness: - Leber’s congenital amaurosis – rAAV vectors 3. Hemophilia B – rAAV-8 vectors; liver-targeted delivery 4. Metabolic diseases - lipoprotein lipase deficiency - first registered drug

Some beneficial effects have been observed in treatment of:

1. – lentiviral vector & hematopoietic stem cells 2.β-thalassemia – lentiviral vector & hematopoietic stem cells

52

Examples of gene therapy strategies undertaken at the Department of Medical Biotechnology Blood vessel formation

Vasculogenesis in embryo Physiological angiogenesis in adults

placenta uterus

Wound healing Hair growth Different phases of skin repair

Inflammatory phase

New tissue formation

Remodelling phase

Schafer & Werner, Nature Review Mol Cell Biol 2008 New capillary formation in response to wounding Clinically relevant healing impairment in genetically diabetic mice - leptin receptor deficient (db/db)

• obesity • insulin resistance dbdb WT • severe hyperglycemia (that resembles human adult onset diabetes) • markedly delayed wound healing

100

80 WT db/db 60 Wound closure in diabetic (dbdb) 40 and normoglycemic (WT) 20 mice % % size of wound the initial 0 0 1 2 3 4 5 6 7 8 10 11 13 15 days after wounding New capillary formation in response to wounding

Heme is released during skin injury and induces HO-1 expression

Wagener et al, Blood, 2003 Hanselmann et al.., 2001 Functions of heme oxygenase-1

PRODUCT MECHANISM ACTIVITY

BILIVERDIN antioxidant anti-inflammatory ROS scavenging BVR ROS anti-apoptotic Inhibition of complement pro-angiogenic BILIRUBIN cytoprotection

ferritin antioxidant HO-1 synthesis anti-inflammatory HEME Fe2+ iron ATP-ase anti-apoptotic pump cytoprotection

activation of sGC leading to cGMP production anti-apoptotic anti-proliferative p38 MAPK regulation anti-thrombotic CO anti-inflammatory pro-angiogenic others cytoprotection

Reviewed in: Dulak et al. Circulation 2008:, 117: 231-241 Expression of HO-1 in wounded skin

Day 0 Day 10

Western blot tubulin

HO-1 H 1 2 3 5 8 11 14 21 PC Days after wounding

Grochot-Przeczek et al., PLoS One. 2009;4(6):e5803 Impaired wound healing in diabetic mice

WT

db/db

Grochot-Przeczek et al. , PLoS ONE 4(6): e5803; 2009 Expression of HO-1 in diabetic mice

tubulin

HO-1

days after 0 1 3 8 17 1 3 8 17 wounding

WT db/db wounded skin

Grochot-Przeczek et al., PLoS One. 2009;4(6):e5803 Neovascularization of the wounded skin in diabetic mice db/db WT

Day 0 40 WT * * db/db

surface 30 Day 1 per * ] 20 Day 3 vessels # * units * # # 10 #

Day 8 umber of arbitrary [ N 0 0 1 3 8 17

Days after wounding Day 17

CD31 expression

Grochot-Przeczek et al., PLoS One. 2009;4(6):e5803 Expression of HO-1 and GFP transgenes in the skin

AdGFP AdHO-1

Grochot-Przeczek et al., PLoS One. 2009;4(6):e5803 Neovascularization of the in diabetic mice injected with AdHO-1

Alkaline phosphatase, CD31 expression, 14 ** AdGFP 3 days after wounding 3 days after wounding

12 AdHO-1

10

8 AdGFP 6 [arbitrary units] 4

Number of vessels vessels ofNumber per surface 2

0 AdHO-1 3 14 Days after wounding

Grochot-Przeczek et al., PLoS One. 2009;4(6):e5803 Adenoviral gene therapy with HO-1 accelerates early phases of wound healing

Grochot-Przeczek et al. , PLoS ONE 4(6): e5803; 2009 Conclusions

- HO-1 improves cutaneous wound healing, which is possibly associated with increased angiogenesis.

- Impaired induction of HO-1 in wounded skin appears to contribute to the delayed wound healing in diabetic mice.

- Overexpression of HO-1 by gene transfer may facilitate wound healing in diabetic mice.

Post-ischemic blood flow recovery and neovascularization is impaired in HO-1 deficient mice

Jazwa A et al., Cardiovasc Res, 2013: 97:115-24 Hypoxia-inducible factors

O2 HYPOXIA NORMOXIA PROLYL HYDROXYLASES HIF-1α HIF-1α O2 Fe2+ CO2 OH OH 2-OXOGLUTARATE SUCCINATE pVHL stabilization

proteasomal degradation Hypoxia-driven hHO-1 gene therapy

O HIF1α 2 proteasomal degradation CO p38 hypoxia/ Fe2+ ferritin ischemia BV/BR Stabilization HO1 anti-inflammatory HIF1α heme anti-oxidative anti-thrombotic Nucleus anti-apoptotic

HIF1α HIF1β HRE minCMV HO-1 cDNA ischemic tissue protection Jaźwa et al., Biotechnologia 2011 3x Human HO1 pHRE-HO-1 vector

Human endothelial cells

Jazwa A et al., Cardiovasc Res, 2013: 97:115-24 Local overexpression of HO-1 improves perfusion in ischemic hindlimbs o i 1.25 t pHRE-empty a r pHRE-HO1 c i 1.00 m * e w o l 0.75 f s c h i d - o n o o

l 0.50 n B c / i

m 0.25 e s c h i 0.00 day 0 day 7 day 14

pHRE-empty pHRE-HO1

Capillaries in adductor muscle 0 y a d 4 1 y a d

min max

Jazwa A et al., Cardiovasc Res, 2013: 97:115-24 HRE-driven HO-1 attenuates inflammation and enhances muscle regeneration

Jazwa A et al., Cardiovasc Res, 2013: 97:115-24 Texts to read! More complex (and better?) approach

Hindlimb ischemia O post-ischemic blood flow HIF-1α 2 proteasomal degradation HO-1 TLR-mediated inflammation Stabilization regenerative potential of muscles in hypoxia

HIF-1α post-ischemic blood flow inflammation nucleus VEGF HIF-1α HIF-1β Expression pHRE-HO-1-VEGF plasmid vector 3xHRE minCMV HO-1 cDNA IRES VEGF cDNA

endogenous HIF-regulated gene expression

Jazwa et al., Vascular Pharmacol 2015 Combined HO-1 and VEGF gene therapy provides better revascularisation than single gene

Jazwa et al., Vascular Pharmacol 2015 Role of HO-1 in cardiovascular system

Induction (injury, pathogens, modified lipids. ROS… )

Hmox1 expression

HO-1 enzyme HO-1 protein

Fe2+ CO Biliverdin/bilirubin Modulation of transcription factors

Atherosclerosis Endothelial protection Diabetes Angiogenesis Myocardial infarction Hypertension Stem cells differentiation Hind limb ischemia Wound healing Innate & acquired immunity Restenosis Transplantation Reviewed in: Dulak et al. – special issue of Antioxid Redox Signal, 2014 Text to read! Exam – planned on 1st February (Monday), room D107, 1 pm