Post on 03-Feb-2022
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Microbubbles and ultrasound for gene therapy
Martin BlomleyImaging Sciences Department
Faculty of Medicine, Imperial CollegeRadiology, Hammersmith Hospital
London
Mayneord Philips Summer SchoolJuly 2005 Oxford
Microbubble ultrasound for gene therapy
• Basic principles• What does the in vitro data tell us?• Recent in vivo studies• How near to clinical applications?
Clinical trials in gene therapy:
www.wiley.co.uk/genmedRetrovirus
34%
Adenovirus27%
Poxvirus6%
Adeno-associated virus2%
HSV1%
Others2%
N/C4%
Lipofection12%
Naked DNA11%
RNA transfer1%
Safe efficient site specific delivery: Viral
• Pro:– Better transfer efficiency
• Con:– Immunogenicity (especially adenovirus)– Cytopathic effects (especially herpes)– Undesirable viral tropisms– Limitations on the length of DNA that can be
carried
Safe efficient site specific delivery: Non-viral
• Pro:– Ease of preparation– Better safety– Less immunogenicity and inflammatory side effects– Can carry relatively large DNA sequences
• Con:– Efficiency and target precision poor even when
complexed– Short duration of effect
Ultrasound potentiatestransfection
Fechheimer M et al. Eur J Cell Biol 1986;40(2):242-7 and PNAS 1987: 84: 8463-7
Manome Y et al. Human Gene Therapy 2000; 11: 1521-8Miller DL et al. Ultrason Med Biol 1999; 25(9):1425-30.Huber PE, Pfisterer P. Gene Therapy 2000; 7: 1516-25.
Ultrasound
Can create pores in cell membranes“Sonoporation”
Generally higher powerthan FDA limits for diagnostic use
Tachibana et al Lancet 1999
Method
US
1. FITC-dextran 5mins2. Wash
Time
“Negative control”: No ultrasound“Positive control” : Already in contact
with FITC when US applied
4 kDa FITC-dextranRMFI
0
1
2
3
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5
6
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9
Positivecontrol
Negative(no US)
10 sec 30 sec 10 mins 30 mins 60 mins 180mins
270mins
4KDa FITC-dextran :early time points
RMFI
0
2
4
6
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10
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20
5 mins before 1 min before Just before Just after 2 secondsafter
5 secondsafter
Control
US
Effects of US power
4ug DNA 20S
Effects of exposure duration
2W/cm2, 4ug DNA
Cell death increases with power
0
10
20
30
40
50
60
0 0.5 1 1.5 2 2.5 3 3.5
Power W/cm2
0
10
20
30
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60
70
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90
100
0 10 20 30 40 50 60 70
Exposure / s
Cell death increases with exposure duration
QuickTime™ and aCinepak decompressor
are needed to see this picture.
Kathy Ferrara and colleagues, UC Davis
Microbubbles: efficient promotorsof ultrasound bioeffects
Porter T et al. J Ultrasound Med 1996 Aug;15(8):577-84
Miller D and Quddus J. Ultrasound Med Biol 2000; 2694): 661-7Greenleaf W et al. Ultrasound Med Biol 1998: 2494): 587-595.Lawrie A et al. Circulation 1999; 99: 2617-20.Porter T et al. Ultrasound Med Biol 2001 Feb;27(2):259-65Shohet R et al. Circulation 2000 6;101(22):2554-6
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Microbubbles could enhance extravascular delivery
•• TumourTumour
•• CapillariesCapillaries
•• RBCRBC
•• MicrobubblesMicrobubbles
••Bursting bubblesBursting bubbles
••Released Released drugdrug
•• UltrasoundUltrasound
Price, et al., Circulation, 1998;98:1264-1267
Tissue specific microbubbles
Blomley MJKet al. Stimulated acoustic emission in the liver parenchyma with the ultrasound contrast agent Levovist. Lancet 1998 ; 351(9102): 568Blomley MJK et al. Improved imaging of liver metastases in the late enhancement phase of the ultrasound contrast agent Levovist,. Radiology 1999: 210(2): 409-16Albrecht T, Blomley M, Burns P et al. Improved detection of liver metastases during the liver phase of SHU508A. Radiology 2003; 227(2):361-70.
Tissue specific microbubbles
Blomley MJKet al. Stimulated acoustic emission in the liver parenchyma with the ultrasound contrast agent Levovist. Lancet 1998 ; 351(9102): 568Blomley MJK et al. Improved imaging of liver metastases in the late enhancement phase of the ultrasound contrast agent Levovist,. Radiology 1999: 210(2): 409-16Albrecht T, Blomley M, Burns P et al. Improved detection of liver metastases during the liver phase of SHU508A. Radiology 2003; 227(2):361-70.
Lim et al. Radiology 2004; paper in press
Targeted/preloaded microbubbles
After Unger
Targeted microbubbles• Microbubbles which target P-selectin and αv-
integrins (Klibanov, Linder and colleagues)• Thrombus specific microbubbles which target
GPIIb/IIIa (Unger, Schneider and colleagues)• Schumann PA et al. Invest Radiol 2002:27(11):587-93
Precontrast PostcontrastPrecontrast Postcontrast
No-invasive assesssment of angiogenesis by US and microbubbles targeted to αv integrins. Leong-Poi…Lindner. Circulation 2003; 107: 455-60
Antibody-labelled microbubbles targeting to cells
Microbubbles as site-specific delivery vehicles
after Unger
0
20
40
60
80
Number of GFP positive fibres
DNAonly
DNA+US
DNA+MB
DNA+MB+US
DNAonly
DNA+US
DNA+MB
DNA+MB+US
0
1
2
3
4
5
6
Maximum area (mm2)
• Plasmid only Plasmid/OptisonPlasmid/Optison/US
0
25
50
75
100Number of GFP positive fibres
DNAonly
DNA+US
DNA+MB
DNA+MB+US
0
2
4
6
8Maximum area (mm2)
DNAonly
DNA+US
DNA+MB
DNA+MB+US
DNAonly
DNA+US
DNA+MB
DNA+MB+US
0
5
10
15Units of GFP intensity
• Plasmid DNA only
• Plasmid/US
• Plasmid/microbubbles
• Plasmid/microbubbles/US
An Extra digit(#3) produced by SonoporationGene Induction
Oligonucleotides
Evidence for:• potentiation of transfer with US• binding to some microbubbles
Oligonucleotides showing considerable promise especially in gene modulation / silencing strategies
• antisense• decoy strategies• RNA interference
Recent oligo studies
Erikson et al. Mol and Cellular Cardiol 2003• Antisense to TNF-α in the heart with
microbubble ultrasound: downregulatedAzuma et al. Gene Therapy 2003. • Decoy to NFκ-B: improved delivery with
microbubble ultrasound and improved survival of rat allografts
How near are we to clinical use?Ultrasound widely used and available and safeMicrobubbles are also available clinically in many
countriesDefinite promise as a non-viral delivery tool for
genes and oligonucleotidesBioeffects of microbubble ultrasound do need more
evaluationSubstantial development work still needed for
therapeutic applications