CANADIAN RESEARCH FOCUS
April 2, 2010

Semple, S., Akinc, A., MacLachlan, I. et al. just published a new study
"Rational design of cationic lipids for siRNA delivery”, Nature Biotechnology (2010), Feb;28(2):172-176.

Interview with author, Dr. Ian MacLachlan

Dr.MacLachlan received his B.Sc. and Ph.D in Biochemistry from the University of Alberta, and then spent the next two years researching systemic gene transfer at the Vienna Bio-Center, followed by postdoctoral research with Dr.Nabel in the area of DNA-based therapeutics at the Howard Hughes Medical Institute at the University of Michigan.  He was the head of gene transfer technology at Inex Pharmaceuticals (Burnaby, British Columbia) before moving to Protiva in 2000 where he led the R&D program until the merge with Tekmira in 2008.  Dr. MacLachlan is currently the executive vice president and chief scientific officer at Tekmira Pharmaceuticals (Burnaby, British Columbia) as well as a member of the New York Academy of Sciences, the Oligonucleotide Therapeutics Society, and the American Society of Gene Therapy.

Dr. MacLachlan's research is currently focused on molecular therapeutics.  His research team consists of more than 25 scientists specializing in the areas of biochemistry, molecular biology, immunology, pharmacology, and lipid chemistry.

Nucleic acid delivery by traditional methods is limited as these large molecules are relatively unstable in the bloodstream and cannot diffuse readily across the membranes of target cells.  In particular, siRNA with it high molecular weight, anionic charge and hydrophilicity cannot passively diffuse across most cell plasma membranes.  Tekmira has recently developed a siRNA delivery system known as “SNALP” (stable nucleic acid-lipid particles) in which a variety of nucleic acids including siRNA can be encapsulated and systemically delivered with specialized lipid nanoparticles.  The following figure (Figure 1) shows SNALP as designed by Tekmira.

As these nucleic acid encapsulated particles can circulate for a long time in the blood, the SNALP technology increases the accumulation at sites of vascular leak (i.e. tumor cell growth, infection or inflammation).  Target cells then take up the SNALP through endocytosis and the nucleic acid is successfully delivered inside cell.  The following figure (Figure 2) shows the delivery of siRNA using SNALP.

In his latest article in Nature Biotechnology, Dr. MacLachlan and his research team report on the ability of cationic lipids to be designed for the delivery of small interfering RNA.  The study successfully achieved the silencing of a therapeutically significant gene which represents a substantial improvement in activity shown in prior report on lipid nanoparticles–siRNA mediated silencing.

CCCRS:
Regarding the siRNA delivery, can you explain the main differences technically between delivery of si-RNA vs DNA? 

Dr. Ian MacLachlan:
Chemically, siRNA and DNA are very similar, but biologically, the two are very different, especially when we consider expressible DNA constructs such as plasmid DNA. Plasmids require delivery to the cell nucleus and the nuclear envelope presents an almost insurmountable obstacle. Delivery of small siRNA, which acts in the cytoplasm, is relatively efficient. Plasmid DNA is typically prepared from fermented bacterial cell cultures. As a bacterially derived biologic, plasmids contain immune stimulatory unmethylated CpG motifs which can act to limit the tolerability of plasmid DNA and confound the interpretation of pre-clinical results. Synthetic siRNA chemistries can be exquisitely controlled allowing for the development of safe and effective siRNA-based drugs with a minimum of off target effects.

Though not strictly a technical issue, drugs containing plasmid DNA are regulated by the FDA's Center for Biologic Evaluation and Research (CBER) while synthetic siRNA based drugs are evaluated by the Center for Drug Evaluation and Research (CDER). Expressed plasmid DNA is also subject to regulation by the National Institute of Health's Office of Biotechnology Activities Recombinant DNA Advisory Committee, a second level of regulatory oversight which is though by many to be an additional regulatory burden unduly encumbering the development of gene based drugs. Synthetic siRNA are not subject to this level of oversight.

CCCRS:
How quickly does siRNA typically take to silence a targeted gene and how long does it last? 

Dr. Ian MacLachlan:
siRNAs can be very fast acting, achieving 50% of maximal silencing levels within one hour after administration of a single dose and maximal silencing 24-48 hours later. A single dose of siRNA delivered using Tekmira's SNALP can silence the target transcript for more than 30 days in both mice and non-human primates.

CCCRS:
What is currently the best delivery efficiency that can be obtained?

Dr. Ian MacLachlan:
We can silence the expression of hepatocellular target transcripts, such as ApoB, by greater than 95% in vivo. Our understanding is that this represents efficient delivery to all the cells in the liver (approximately 100%) with some level of heterogeneity in terms of the degree of silencing in individual subpopulations of cells.

CCCRS:
In your paper you describe the design of a cationic lipid comprised of a head group, linker and hydrocarbon chain.  The paper concentrates on the linker and head group chemistry.  What is the effect of the hydrocarbon chain length?

Dr. Ian MacLachlan:
Cationic lipid hydrocarbon chain length will influence membrane fluidity and presentation of the cationic head group (charge). Other than that we haven't disclosed the effect of hydrocarbon chain length.

CCCRS:
You use pKa and LII to HII transition temperature at pH 4.8 to screen chemistries for having no charge in blood and less non-specific membrane disruption and endosome bilayer disruption potential.  Your in vivo work showed that this was not always effective in predicting efficacy.  Were you surprised by this and what further in vitro tests might help identify lead candidates?

Dr. Ian MacLachlan:
We were not surprised, but I'm not able to comment further.

CCCRS:
How important is the lipid size?  And, you refer to them as being nanosized, how big is that?

Dr. Ian MacLachlan:
The particle size is very important because it influences the biodistribution of the particle. For many applications, the target cells are the hepatocytes of the liver. These cells are best reached by passing through the fenestrae of the liver vasculature and as such the ideal particle size is one which is smaller than the average liver fenestration. Although in 2010 it may seem that efficient delivery of nucleic acids to the hepatocytes is straightforward, this has not always been the case. It is only recently, through the use of newly developed manufacturing methods and highly fusogenic polyunsaturated titratable aminolipids, such as those described in our recent paper, that efficient delivery of nucleic acids to the hepatocytes, bypassing the cells of the reticuloendothelial system, has been realized.

Our SNALP particles are typically 80 nm in diameter, but can be manufactured anywhere from 40-140 nm in diameter.

CCCRS:
How important is lipid loading efficiency? And, is this affected by lipid design?

Dr. Ian MacLachlan:
Loading efficiency is very important as siRNA and other nucleic acids are quite costly relative to conventional small molecule drugs. Our encapsulation efficiencies range from about 80 % to 95 %, depending on the scale, with the highest encapsulation efficiencies achieved at the higher manufacturing scales.

Lipid design is one critical parameter in determining encapsulation efficiency, second only to the manufacturing method itself. 

CCCRS:
What is the major hurdle to be overcome before patients can benefit from this technology?  And, what are the potential dangers? 

Dr. Ian MacLachlan:
The major hurdle remaining is the demonstration of safety and efficacy in human clinical trials. We have done everything we can in the laboratory to de-risk the technology but it's the clinical experience that counts.

One potential danger is that in our rush to be among the first to develop drugs using this technology, we rush or otherwise cut corners in ways that compromise patient safety. We can't allow that to happen.

CCCRS:
Could you predict when siRNA therapies will be available?

Dr. Ian MacLachlan:
I expect that there will be siRNA-based drugs on the market within the next 5 or 6 years.

CCCRS:
Thank you for the interview!