To reviewer #1:
Comments of reviewer #1 (1)
The authors describe nanoparticles which contain a DNA core packaged into a lipid
envelope modified with an octaarginine peptide. The peptide mediates internalization
via macropinocytosis which avoids lysosomal degradation. Topical application of the
DNA nanoparticles containing GFP or other genes resulted in their expression in the
hair follicle. The results are interesting.
Answer to reviewer #1’s comment (1)
We would like to thank the reviewer for highly evaluating our gene delivery system.
Comments of reviewer #1 (2)
However, the authors ignore the pioneering work on hair-follicle gene therapy (Nature
Medicine 1, 705-706, 1995; Proc. Natl. Acad. Sci USA 99, 13120-13124, 2002) which
calls into question the degree of novelty of their work. The first paper cited above
demonstrates specific hair follicle targeting of genes, a topic not discussed sufficiently
in the present manuscript. The latter paper cited above used gene therapy to change the
phenotype of the hair shaft itself, which was not achieved in the present study.
Answer to reviewer #1’s comment (2)
As reviewer #1 pointed out, Li and Hoffman succeeded in gene delivery to hair
follicles using liposome-based non-viral delivery system, and Saito et al. reported gene
delivery to hair follicles of mouse skin fragments by adenovirus vector. We agree with
the reviewer’s criticism that we should have cited these papers. In addition, we also do
not expect that there is a novelty in the gene delivery to hair follicles. We believe that
the novelty in our manuscript lies in the higher transgene ability to hair follicle of our
R8-MEND than the well known transfection reagent Lipofectamine and hair cycle
control by delivery of a functional gene (BMPR1A). Thus, according to reviewer #1’s
comment, we cited the two papers suggested, and added a brief description of their
works to our new manuscript as follows;
“The topical delivery of genes to hair follicles (HFs) is an attractive approach for the
treatment of skin and hair disorders21-23. Li and Hoffman succeeded in delivery of lacZ
gene to mouse hair-forming hair matrix cells in the hair follicle bulbs using
liposome-based non-viral delivery system21, and Saito et al. reported gene delivery to
hair follicles of mouse skin fragments by adenovirus vector22. Domachenko et al. also
reported delivery of lacZ gene to hair follicle progenitor cells of mouse and human by
liposome-based non-viral system23.” (page 11, lines 10-15)
References in this paragraph;
Ref No.21: Li L, Hoffman RM. The feasibility of targeted selective gene therapy of the
hair follicle. Nat Med 1995; 1: 705-706. (Newly added reference)
Ref No.22: Saito N, Zhao M, Li L, Baranov E, Yang M, Ohta Y, Katsuoka K, Penman S,
Hoffman RM. High efficiency genetic modification of hair follicles and growing hair
shafts. Proc Natl Acad Sci USA 2002; 99: 13120-13124. (Newly added reference)
Ref No.23: Domashenko A, Gupta S, Cotsarelis G. Efficient delivery of transgenes to
human hair follicle progenitor cells using topical lipoplex. Nat Biotechnol 2000; 18:
420-423.
Comments of reviewer #1(3)
The authors also do not cite the development of GFP for imaging gene transfer in vivo
(Proc. Natl. Acad. Sci. USA 97, 12278-12282, 2000), a technique the authors relied on.
It is important that this manuscript be re-written in the context of the existing
knowledge of hair follicle gene therapy.
Answer to reviewer #1’s comment (3)
According to reviewer #1’s comment, we cited the paper about in vivo imaging
technique using GFP gene suggested by reviewer #1, and added a description of that
paper to our new manuscript as follows;
“The use of green fluorescence protein (GFP) reporter gene has been established as a
marker for visualizing gene expression at different tissues in the whole body by Yang et
al.24. MEND3 particles containing a GFP reporter gene produced similar results as
MEND3-LacZ, with GFP fluorescence occurring in treated tissue two weeks after
treatment with MEND3-GFP (Supplementary Fig. 2 on line).” (page 12, lines 8-11)
References in this paragraph;
Ref No.24: Yang M, Baranov E, Moossa AR, Penman S, Hoffman RM, Visualizing gene
expression by whole-body fluorescence imaging. Proc Natl Acad Sci USA 2000; 97:
12278-12282. (Newly added reference)
To reviewer #3:
Comments of reviewer #3 (1)
This manuscript reported an interesting result of MEND, which is comparable to
adenovirus as gene delivery system. The manuscript was clearly presented and well
organized.
Answer to reviewer #3’s comment (1)
We are grateful for the reviewer for his supportive opinion about the clarity and
organization of our manuscript.
Comments of reviewer #3 (2)
The author should discuss on the size effect of MEND1-3, since their sizes are different
each other.
Answer to reviewer #3’s comment (2)
Based on the reviewer comment, we discussed the size effect of different MENDs in
the new version of the manuscript as following:
“Although the diameters of MENDs1-3 were somewhat different (Supplementary
Table 1), it is suggested that this difference has no significant effect on the uptake
pathway of the particles. We have previously reported that 5% R8-liposomes of around
100 nm was taken up by macropinocytosis11. Here, we confirmed that the larger
R8-MEND3 particles (330 nm) are taken up mainly by the same pathway (Fig. 3). Even
much smaller arginine-rich peptides and peptide-fusion proteins were taken up by
macropinocytosis30,32. This suggests that the size difference of different MENDs does
no affect the uptake pathway. In addition, the uptake via macropinocytosis involves the
formation of large macropinosomes (>1m) which allows the internalization of small as
well as larger MENDs. This is clearly different form the classical clathrin-mediated
endocytosis (involves endosomes with a size limit of around 150 nm)2, which may not
explain the uptake of MEND3 particles. Regarding the transfection efficiency of
different MENDs, we believe that the superiority of MEND3 is mainly due to the
enhanced fusiogenic ability of the DOPE/CHEMS combination11 rather than due to a
size effect. However, the possibility that larger particles may sediment more on the cell
surface35, thus allowing more internalization, cannot be completely excluded.” (page 19,
line 1- page 20, line 1)
References in this paragraph;
Ref No.11: Khalil IA, Kogure K, Futaki S, Harashima H. High density of octaarginine
stimulates macropinocytosis leading to efficient intracellular trafficking for gene
expression. J Biol Chem 2006; 281: 3544-3551.
Ref No.30: Wadia JS, Stan RV, Dowdy SF. Transducible TAT-HA fusogenic peptide
enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat Med
2004; 10: 310-315.
Ref No.32: Kaplan IM, Wadia JS, Dowdy SF. Cationic TAT peptide transduction
domain enters cells by macropinocytosis. J Control Release 2005; 102: 247-253.
Ref No.2: Khalil IA, Kogure K, Akita H, Harashima H. Uptake pathways and
subsequent intracellular trafficking in non-viral gene delivery. Pharm Rev 2006; 58:
32-45.
Ref No.35: Harris SS, Giorgio TD. Convective flow increases lipoplex delivery rate to
in vitro cellular monolayers. Gene Ther 2005; 12: 512-520. (Newly added reference)