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Tongji Medical College, HUST

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1 Tongji Medical College, HUST
Recombinant Adeno-associated Virus-Mediated Human Kallikrein Gene Therapy of Hypertension Dao Wen Wang The Institute of Hypertension, Tongji Hospital Tongji Medical College, HUST

2 ? ? Gene Therapy of Hypertension Vasodilatror Genes Vasoconstrictor
The Choice of target gene ? Insertion of Gene Copies The Choice of vector Antisense Inhibition ?

3 The Kallikrein-Kinin System
(+) Kinin`s metabolities LMW kininogen Kinin (-) ACE (RAS) IP3-Ca2+ DG-PKC Vasodilatation PLC BK1 receptor BK2 receptor Smooth-muscle relaxation PLA2 cAMP NO cGMP Renal perfusion PGI2 Inflammation

4 Objective Delivery human kallikrein (HK) cDNA in recombinant adeno-associated virus (rAAV) to observe: Long term and stable expression of HK Therapeutic effects on hypertension Mechanisms for kallikrein delivery to attenuate cardiovascular and renal remodeling

5 The rAAV-HK Virus Packaging
pXX2 pXX6 pXXUF1-HK (pdx-lacZ) (1:1:1 mol ration) (Ca-P) 293 cells(150mm plate) hour rAAV-HK Cell harvested Frozen and thawed Purified rAAV-lacZ Dot blot hybridization

6 Part I Intravenous Delivery of rAAV-HK Virus Reduces Hypertension and Attenuates Cardiovascular and Renal Injury in Spontaneously Hypertensive Rats

7 Methods SHR Blood pressure monitoring Expression of HK Cardiovascular
(RT-PCR) (ELISA) rAAV-HK Virus SHR Cardiovascular protection (Sirius red staining) Intravenous delivery 20 weeks (heart weight) (Urine Microalbumin) Renal protection (Sirius Red Staining) (HE staininig)

8 HK expression in tissues
Results The expression of HK in SHR Kidney Lung Spleen Liver Heart Kidney Lung Spleen Liver Heart rAAV-HK rAAV-lacZ HK expression in tissues

9 Urine HK level Serum HK level

10 rAAV-HK reduces blood pressure in SHR

11 rAAV-HK protects the cardiovascular system
heart weight/body weight ratio(%) Heart weight/body weight ration Left ventricular weght/heart weight ratio Left ventricular weight/heart weight ratio(%) rAAV-HK rAAV-lacZ rAAV-HK rAAV-lacZ

12 rAAV-HK attenuates the fibrosis in heart
rAVV-HK rAAV-lacZ

13 rAAV-HK attenuates the fibrosis in aorta
rAVV-HK rAAV-lacZ

14 rAAV-HK protects the renal function
rAAV-HK reduces urine microalbumin

15 rAAV-HK attunuates the renal injury
rAVV-HK rAAV-lacZ

16 rAAV-HK attenuates the fibrosis in kidney
rAVV-HK rAAV-lacZ

17 rAAV-HK prevent stroke in SHR

18 Conclusion of Part I 1. The rAAV vectors resulted in a long term and stable expression of HK 2. The expression of HK induced a long term and stable reduction of blood pressure in SHR 3. HK attenuates the cardiovascular and renal injury in SHR 4. there was no obvious toxic and side effect induced by virus injection in animals

19 Part II Intramuscularly Delivery of rAAV-HK Virus Reduces Hypertension and Attenuates Cardiovascular and Renal Injury in SHR

20 (supersonic detection)
Methods Blood pressure monitoring Expression of HK (RT-PCR) (ELISA) (ELISA) rAAV-HK Virus SHR (Sirius red staining) Cardiovascular protection (heart weight) Intramuscularly delivery 24Weeks (supersonic detection) Renal protection (Sirius Red Staining) (HE staininig)

21 The expression of rAAV-HK in SHR
Results The expression of rAAV-HK in SHR

22 Urine HK level Serum HK level

23 Intramusclarly delivery rAAV-HK reduces blood pressure in SHR

24 Intramuscularly delivery of rAAV-HK Protects the renal function in SHR
rAAV-HK attunuates the renal injury rAAV-HK attenuates the fibrosis in kidney rAAV-HK rAAV-lacZ rAAV-HK rAAV-lacZ

25 rAAV-HK attenuates the fibrosis in cardiovascular system
Intramuscularly delivery of rAAV-HK Protects the cardiovascular function in SHR rAAV-HK attenuates the fibrosis in cardiovascular system left ventricular weight/heart weight ratio heart weight/body weight ratio rAAV-HK rAAV-lacZ

26 rAAV-HK attenuates the heart function in SHR
Intraventricular septum(IVS) Fractration shorting(FS%)

27 Skeletal muscle could be preferring target organ for gene therapy
Conclusion of Part II Intramuscular delivery of rAAV-HK Virus also resulted in a long term and stable expression of HK and permanent control of high blood pressure as well as protection of organs in SHR Skeletal muscle could be preferring target organ for gene therapy

28 Part III The Effect of rAAV-HK Virus Delivery on Basal Level of Blood Pressure and High Salt Diet Induced Hypertension

29 Methods SD rats Blood pressure monitoring Expression of HK Renal
rAAV-HK Virus SD rats High salt diet Expression of HK (RT-PCR) (ELISA) Intravenously delivery 5Weeks 8Weeks (urine volume) Renal protection (Na+ K+ excretion) (urinary osmotic pressure )

30 The effect of rAAV-HK virus on blood pressure in SD rats

31 Urine HK level in SD rats
Results The expression of rAAV-HK in SD rats Urine HK level in SD rats

32 The Conclusion of Part III
rAAV-HK injection does not have obviouseffects on blood pressure in SD rats rAAV-HK injection could prevent the increase of blood pressure and attenuate the renal injury induced by high salt intake

33 Part IV rAAV-HK attenuated the end-stage organ remodeling in hypertension through promoting the cell proliferation and inhibiting the cell apoptosis

34 TNF MAPK PI3K AKT Bcl-2 Bcl-xl Caspase 3 Bax

35 Experiment on Animals Methods Tissue Level TUNEL Staining SHR tissues
from PART I Cell activation signaling MAPK, PI3K/AKT Molecular mechanism Antiapoptotic signaling Bcl-2, Bcl-xl Western Blot Proapoptotic signaling Bax

36 Results rAAV-HK inhibiting apoptosis in tissues of SHR
Heart Results rAAV-HK inhibiting apoptosis in tissues of SHR Kidney Aorta rAAV-HK rAAV-lacZ

37 Cell activation related sigaling
MAPK P-MAPK AKT P-AKT β－actin PI3K Heart Kidney Aorta Heart Kidney Aorta rAAV-lacZ rAAV-HK

38 Apoptosis related signaling
β－actin Bcl-2 Bcl-xl Bax Heart Kidney Aorta Heart Kidney aorta rAAV-lacZ rAAV-HK

39 (Flow cytometry assay)
Experiment on Cells Methods Cell activation (MTT) Bradykinin (BK) (AO/EB staining) Apoptosis cell rate BK+Hoe 140 (Flow cytometry assay) 293 cells Apoptotic transduction pathway rAAV-HK (caspase 3 activation) TNF+ActD rAAV-lacZ Apoptosis related signaling (western blot)

40 Results BK, rAAV-HK promoted the cell proliferation (MTT) TNF-α
－ Control rAAV-lacZ TNF- α BK BK+Hoe rAAV-HK

41 Control － TNF ＋ BK ＋ BK+HOE ＋ rAAV-HK ＋ rAAV-lacZ ＋

42 BK, rAAV-HK reduced the apoptotic cell rate(FACS)
TNF-α － Control BK+Hoe rAAV-HK rAAV-lacZ TNF- α BK

43 BK, rAAV-HK reduced the caspase 3 activation in cells
TNF-α － rAAV-lacZ Control BK+Hoe rAAV-HK TNF-α BK

44 Cell activation related signaling
MPAK P-MPAK AKT P-AKT β-actin PI3K － ＋ ＋ ＋ ＋ ＋ Control TNF BK BK+Hoe rAAV-lacZ rAAV-HK

45 Apoptosis related signaling
β-actin Bcl-2 Bcl-xl Bax － ＋ ＋ ＋ ＋ ＋ Control TNF BK BK+Hoe rAAV-lacZ rAAV-HK

46 Conclusion of part IV rAAV-HK attenuated the end-stage tissue remodeling through promoting kidney epithelial cell proliferation and inhibiting apoptosis rAAV-HK could promote the cell proliferation by activating PI3K/Akt and MAPK pathways rAAV-HK could inhibited the cell apoptosis by down-regulating the production of Bax and up-regulating the Bcl-2 and Bcl-XL production

47 Conclusions rAAV-mediated delivery of HK gene in vivo through both Intravenous and Intramuscular ways resulted in a long term and stable expression of HK, and reduced blood pressure as well as attenuated cardiovascular and renal injuries in SHR. rAAV-HK could inhibit the increasing of blood pressure induced by high salt intake and protect the renal function in it, but not affect the normal blood pressure.

48 It is a promising gene drug for hypertension therapy
rAAV-HK could inhibit the increasing of blood pressure induced by high salt intake and protect the renal function in it. rAAV-HK and bradykini attenuated the end-stage tissue remodeling through promoting cell activation and inhabiting apoptosis through promoting the cell proliferation and inhibiting the cell apoptosis It is a promising gene drug for hypertension therapy

49 THANKS

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