Genetic Engineering of Vein Grafts Resistant to Atherosclerosis

1
Genetic Engineering of Vein Grafts Resistant to
Atherosclerosis

• Kylee Crittenden
• Despina Prasinos
• Plant Biology 450
• November 6th 2008

2
Treatment of Arterial Occlusive Disease

• Disease characterized by the obstruction or
  blockage of the peripheral arteries caused by
  high cholesterol levels, infection, blood clot,
  and diabetes.

(University of Iowa Hospitals and Clinics, 2008)
3
Treatment Coronary Artery Bypass Graft

• Treatment Bypass vein grafting using a vein from
  another part of the body to circumvent the
  obstructed blood vessel.

http//www.surgeryencyclopedia.com/Ce-Fi/Coronary-
Artery-Bypass-Graft-Surgery.html
4
Coronary Artery Bypass Graft

• According to the American Heart Association,
  469,000 coronary artery bypass graft operations
  were done in the United States in 2005.
• 325,000 men
• 145,000 women

• 100,000 lower extremity bypass procedures
  performed a year

5
Problem with Treatment Accelerated
Atherosclerosis

• Up to 50 of vein bypass grafts fail with in a
  period of 10 years.
• Why?
• Accelerated atherosclerosis associated with
  neointimal hyperplasia of vein graft.

Mann et. al., 1995
6
Hyperplasia vs. Hypertrophy

• Neointimal Hyperplasia vs. Vascular Hypertrophy
  (http//www.merriam-webster.com/)
• Neointimal Hyperplasia- a new or thickened layer
  of arterial intima formed by cell proliferation
• Vascular hypertrophy- an increase of the size of
  arterial wall due to an increase in the size of
  cells, while the number stays the same

http//commons.wikimedia.org/wiki/ImageHypertroph
y.jpg
7
Neointimal Hyperplasia
http//www.temple.edu/medicine/faculty/a/autierim.
asp?pms(autieri20MV5Bau5D20Temple20Universit
y5Baffiliation5D)
8
Problems cont.

• Injury due to surgery and shear stress to the
  vessel wall induces neointimal hyperplasia.
• Although neointimal hyperplasia that occurs in
  vein grafts provides mechanical stability, it is
  believed that this neointima serves as the
  substrate for the development of atherosclerotic
  disease.
• Vascular hypertrophy still provides strength to
  the vessels but without the increased risk of
  atherosclerosis.
• Atherosclerosis YouTube video
• (Mann et. al., 1995)

9
Solution Genetic engineering of vein grafts that
are resistant to atherosclerosis

• Genetic engineering of vein grafts to increase
  vessel wall via vascular hypertrophy rather than
  hyperplasia.
• Promote cell growth but inhibit cell division.
  
• 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
• Antisense oligodeoxynucleotide blockade of
  expression of the genes for two cell cycle
  regulatory proteins.
• Cell division cycle 2 kinase (cdc2 kinase) and
  proliferating cell nuclear antigen (PCNA)
• Blocks the gene expression necessary for
  transition of smooth muscle cells to the
  replication phase that permits hypertrophy but
  inhibits the proliferation in neointimal
  hyperplasia.
• (Mann et. al., 1995)

10
Methods

• Rabbit vascular smooth muscle cells (SMCs) were
  treated with hemagglutinating virus of Japan
  (HVJ) liposome containing the antisense ODN (in
  vitro transfection).
• The transfected SMCs were directly injected into
  the rabbits distal vein (in vivo transfection).
• Vein graft was performed to introduce the
  transfected distal vein for the jugular vein.
• (Mann et. al., 1995)

11
Results and Discussion

• HVJ-Liposome in vivo transfection technique has
  been found to be highly efficient in delivering
  antisense ODN to the vascular wall.
• Transfection of medial SMCs with antisense ODN
  targeted against PCNA and cdc2 kinase in
  combination has successfully prevented the cell
  cycle progression necessary for neointimal
  hyperplasia in an arterial model.
• (Mann et. al., 1995)

12
Results and Discussion Cont.

• This technique demonstrated that by blocking
  neointimal hyperplasia during the initial
  postoperative period, when the graft is
  responding both to the stresses of the surgery,
  the graft can be pushed to adapt to these
  arterial stresses by medial hypertrophy instead.
• The vein grafts developed medial hypertrophy
  which still allowed the thickening of the vein
  wall to withstand pressure that is necessary to
  deliver blood to the lower extremities.
• No plaque formation was observed in any of the
  antisense-treated grafts in animals fed
  cholesterol diets.
• The antisense ODN- treated grafts proved
  resistant to the formation of diet-induced
  atherosclerotic plaque.
• The arrest of smooth muscle cell (SMC)
  proliferation inhibited neointima formation for
  up to 10 weeks after surgery.
• (Mann et. al., 1995)

13
Further Research E2F Decoy Strategy

• Long-term stabilization of vein graft wall
  architecture and prolonged resistance to
  experimental atherosclerosis after E2F decoy
  oligonucleotide gene therapy.
• The transcription factor E2F is associated with
  upregulation of expression of a dozen genes
  involved in DNA synthesis and cell-cycle
  progression.
• Free E2F can be blocked from binding DNA, thus
  preventing the upregulation of multiple
  cell-cycle genes in vascular cells.
• use ds ODN having the consensus sequence of E2F
  binding site
• (Ehsan et. al., 2001)

14
(Ehsan et. al., 2001, p. 715)
15
Further Research E2F Decoy Strategy

• E2F decoy redirected the graft biology away from
  neointimal hyperplasia toward medial hypertrophy
  and provided long term resistance to
  atherosclerotic disease.
• Previous technique only allowed for 10 weeks of
  resistance while the E2F strategy still provided
  resistance at 6 months.
• This technique is achieved ex vivo without the
  use of liposomes or viral vectors.
• May be a safer approach than previous methods for
  application to human bypass vein grafts

(Ehsan et. al., 2001)
16
Phase I Testing

• The only human studies on vein bypass healing to
  date have used the E2F-decoy strategy
• PRoject of Ex-vivo Vein graft ENgineering via
  Transfection (PREVENT)
• 41 patients were randomly assigned untreated
  (16), E2F-decoy-treated (17), or
  scrambled-oligodeoxynucleotide-treated (8) human
  infrainguinal vein grafts.
• Groups did not differ for postoperative
  complication rates.
• At 12 months fewer graft occlusions were seen in
  the E2F-decoy group than in the untreated group.
• Conclusion
• Intraoperative transfection of human bypass vein
  grafts with E2F-decoy oligodeoxynucleotide is
  safe, feasible, and can achieve sequence-specific
  inhibition of cell-cycle gene expression and DNA
  replication. Application of this
  genetic-engineering strategy may lower failure
  rates of human primary bypass vein grafting.

(Mann et. al, 1999)
17
Phase II Testing

• In 2001, phase II trial was completed in Germany
• As in the phase I trials, no adverse events or
  complications were attributed to the E2F ODN
  treatment.
• The graft level analysis revealed a 30 relative
  reduction in critical stenosis (blood vessel
  narrowing).
• Analysis of intravascular ultrasound scan images
  revealed a statistically significant reduction in
  total wall volume.

(Conte et. al, 2002)
18
Phase III Testing

• In 2003, Phase III testing was conducted with
  1,404 patients
• The data demonstrated that EF2 decoy treatment
  did not significantly reduce the development of
  significant stenoses in a large group of
  patients.
• Additional studies with longer-term follow-up are
  needed to understand the mechanisms and clinical
  consequences of vein graft failure.

(Conte et. al, 2005)
19
References

• Conte, M.S., Bandyk, D.F., Clowes, A.W., Moneta,
  G.L., Seely, L., Lorenz, T.J., Namini, H.,
  Hamdan, A.D., Roddy, S.P., Belkin, M., Berceli,
  S.C., DeMasi, R.J., Samson, R.H., Berman, S.S.,
  and PREVENT III Investigators. (2005). Results of
  PREVENT III A multicenter, randomized trial of
  edifoligide for the prevention of vein graft
  failure in lower extremity bypass surgery.
  Journal of Vascular Surgery, 43(4), 742.
• Conte, M.S., Mann, M.J., Simosa, H.F., Rhynhart,
  K.K., Mulligan, R.C. (2002). Genetic
  interventions for bypass graft disease a review.
  Journal of Vascular Surgery, 36, 1040-1052.
• Ehsan, A. Mann, M.J., DellAcqua, G., Dzau,
  V.J. (2001). Long-term stabilization of vein
  graft wall architecture and prolonged resistance
  to experimental atherosclerosis after E2F decoy
  oligonucleotide gene therapy. Journal of Thoracic
  and Cardiovascular Surgery, 121(4), 714-722.
• Mann, M.J., Gibbons, G.H., Kernoff, R.S., Diet,
  F.P., Tsao, P.S., Cooke, J.P., Kaneda, Y.,
  Dzau, V.J. (1995). Genetic engineering of vein
  grafts resistant to atherosclerosis. Proceedings
  of the National Academy of Sciences of the United
  States of America, 92, 4502-4506.
• Mann, M.J., Gibbons, G.H., Tsao, P.S., von der
  Leyen, H.E., Cooke, J.P., Buitrago, R., Kernoff,
  R., Dzau, V.J. (1997). Cell cycle inhibition
  preserves endothelial function in genetically
  engineered rabbit vein grafts. Journal of
  Clinical Investigation, 99(6), 1295-1301.
• Mann MJ, Whittemore AD, Donaldson MC, Belkin M,
  Conte MS, Polak JF, et al. (1999). Ex-vivo gene
  therapy of human vascular bypass grafts with E2F
  decoy the PREVENT single-centre, randomised,
  controlled trial. Lancet, 354, 1493-1498.
• SoRelle R. (2001). Late-breaking clinical trials
  at the American Heart Associations Scientific
  Session 2001. Circulation, 104(21), 9046.
• University of Iowa Hospitals and Clinics. (2008).
  Arterial Occlusive Disease. Retreived Nov. 2,
  2008, from http//www.uihealthcare.com/topics/card
  iovascularhealth/card3479.html