Supplementary Information

Supercoiled Minivectors resist shear forces associated with gene therapy delivery

DJ Catanese Jr, JM Fogg, DE Schrock II, BE Gilbert and L Zechiedrich

ContentsPage

Supplementary Figure 12

Supplementary Table 13

Supplementary Figure 24

Supplementary References 5

Supplementary Figure 1 Aerosolization of DNA vectors. (a) A Collison-like jet nebulizer1 containing a removable lid that allows for sampling from the reservoir. The nebulizer works as follows: High-pressured air is pumped through a small orifice in the nozzle. The pressure differential between the nozzle and reservoir siphons the therapeutic solution from the reservoir into the high velocity jetstream thus generating primary droplets 15 to 500 m in size.2 Minimal DNA degradation occurs during this primary droplet formation2; however, the droplets are too large to penetrate deep into the lungs.3 A plastic cone within the baffle breaks the primary droplets into smaller (1-10 m) ones that can penetrate deep into the lungs.4 DNA shearing occurs almost exclusively during impact with the plastic cone within the baffle.2 The smaller droplets escape the baffle area and exit the nebulizer in aerosol form. Diagram adapted and reprinted with permission.5 (b) DNA degradation from the aerosol and reservoir for a 3 869 bp plasmid or a 383 bp Minivector. During nebulization, aerosol was captured for 3 min using all glass impingers (AGI) at times 0.5, 7, 20, and 25 min. Each AGI reservoir held 20 mL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). Aerosol output was approximately 0.3 mL min-1. In addition, 15 µL samples were taken from the nebulizer reservoir at times 0 and 28 min. Aerosol and reservoir samples were separated by gel electrophoresis, stained with SYBR®Gold and visualized using PC Image.

Supplemental Table 1 Survival times as a function of DNA length.

DNA length (bp) / a / b / c / Survival90 / Survival75 / Survival25 / Survival10
5 302 / 1.00030 / 1.5263 / 1.8331 / 0.5 / 0.8 / 2.8 / 5.1
3 869 / 0.99002 / 4.1398 / 2.1553 / 1.4 / 2.4 / 6.9 / 11.4
3 000 / 0.99368 / 6.1301 / 2.0214 / 2.0 / 3.5 / 10.5 / 18.1
2 686 / 1.00130 / 10.768 / 3.0935 / 5.3 / 7.6 / 15.4 / 21.9
2 232 / 0.96812 / 16.628 / 3.1301 / 7.3 / 11.2 / 23.3 / 33.2
2 067 / 0.99984 / 19.108 / 3.7264 / 10.6 / 14.2 / 25.7 / 34.5
1 873 / 0.99507 / 22.038 / 3.0363 / 10.5 / 15.3 / 31.6 / 45.4
1 714 / 0.99981 / 28.476 / 2.2510 / 10.7 / 17.5 / 46.4 / 75.6
1 580 / 1.00000 / 35.282 / 2.0585 / 12.1 / 20.7 / 60.2 / 102.6

Data for DNA vectors sheared during nebulization (Figure 1b in main text) were fitted to the sigmoidal equation: y=(a)/(1+[x/b]^c), where y = the fraction of intact DNA; x = time in min; a = normalized upper plateau value, which was a floating variable during the fit; b = Survival50; c = value is reflective of the sigmoidal curve. All survival times are in min. For example, Survival90 (Figure 1c inset in main text)indicates the time at which 90% of intact vectors remain. Data were fitted using Kaleidagraph 4.1.0.

Supplementary Figure 2Nebulizationof plasmids and Minivectors. (a) Competition experiment between a plasmid and four different Minivectors that were subjected to nebulization simultaneously. Additional plasmid was spiked into the reservoir solution at 20 min (depicted by asterisk) to demonstrate that shear forces were still being generated at the later time points. (b) Rate of plasmid shearing over the two additions.

References

1.May KR. The Collison nebulizer: Description, performance and application. J Aerosol Sci 1973; 4: 235-238.

2.Lentz YK, Worden LR, Anchordoquy TJ, Lengsfeld CS. Effect of jet nebulization on DNA: identifying the dominant degradation mechanism and mitigation methods J Aerosol Sci 2005;36: 973-990.

3.Eberl S, Chan HK, Daviskas E, Constable C, Young I. Aerosol deposition and clearance measurement: a novel technique using dynamic SPET. Eur JNucl Med 2001; 28: 1365-1372.

4.Bennett WD, Brown JS, Zeman KL, Hu SC, Scheuch G, Sommerer K. Targeting delivery of aerosols to different lung regions. J Aerosol Med 2002; 15: 179-188.

5.Sambrook J, Russell DW. Protocol 1: Generation of a library of randomly overlapping DNA inserts. In: Argentine J (ed). Molecular Cloning: A LaboratoryManual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York, 2006, pp12.10-12.25.

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