Inicio > Inmunología > “Berlin Patient” and gene therapy against HIV infection

“Berlin Patient” and gene therapy against HIV infection

The case:

Timothy R.B. probably never believed that the same treatment would save him from two different diseases and one of them incurable. He had presented acute myeloid leukemia (FAB M4 subtype) and HIV-1 infection, in the Charité – Universitätsmedizin Berlin (Germany). In the previous 4 years, the patient had been treated with HAART (600 mg of efavirenz, 200 mg of emtricitabine, and 300 mg of tenofovir per day) and no illnesses associated with the acquired immunodeficiency syndrome (AIDS) were observed 1,4,5.

Initial treatment of the acute myeloid leukemia consisted of two courses of induction chemotherapy and one course of consolidation chemotherapy. During the first induction course, severe hepatic toxic effects developed and renal failure occurred. Seven months after presentation, acute myeloid leukemia relapsed, and the patient underwent allogeneic hematopoietic stem-cell transplantation (HSCT) with CD34+ peripheral-blood stem cells from an HLA-identical donor 1,4,5.

Particularly, this donor had been screened for homozygosity for the CCR5 delta32 allele that contains a 32 b.p. deletion, which results in a truncated protein and so generates a non-functional CCR5 receptor phenotype. This chemokine receptor is a cell-surface cofactor for the gp120-CD4 binding, so it is necessary for viral fusion and entry. Some genetic evidence suggests that individuals homozygous for a CCR5 deletion mutation are resistant to HIV infection 1,3,4,5,10,15.

This therapy was described by Eckhard Thiel et al. in “Long-Term Control of HIV by CCR5 Delta32/Delta32 Stem-Cell Transplantation”, on February 12, 2009 in the New England Journal of Medicine 1.

On December 9, 2010 Blood journal pre-published online the article: “Evidence for the cure of HIV infection by CCR5delta32/delta32 stem cell transplantation”, in which the authors showed a reconstitution of CD4+ T cells at the systemic level as well as any sign of HIV infection. The paper quotes: “in conclusion, our results strongly suggest that cure of HIV has been achieved in this patient” 4.

Why gene therapy?

HSCT is used as salvage therapy for patients with malignancies sensitive to chemo-/radiotherapy. These patients receive very dose-intensive cytoreductive regimens designed to eliminate all tumor cells, but the procedure also eliminates the cell substrate necessary to reconstitute the hematopoietic tissue. For this reason it’s require the infusion of HSC (hematopoietic stem cells), that could to reconstitute bone marrow 19.

This therapy presents risks of transplant-related mortality (TRM) due to some complications, as graft-versus-host disease (GvHD), problems derived of inadequate reconstitution of the immune system, the concominant probability of opportunistic infections, and the possibility of malignant relapse. However, the treatment holds great promise as a curative therapy for several tumor types 19, but in the case of HIV infection has insurmountable risks compared with HAART.

Thus, the difficulty in reproducing the therapy is clear, due to the low frequency of the Homozygous Δ32 genotype (1%-3%), the difficulty of finding a matched donor and the problems derived of HSCT. For this reason the application of Gene Therapy seems to be a good option. In this sense I show different approaches that have been made and its possible usefulness in the context of gene therapy against HIV infection.

First of all, I consider myeloablative treatment very important according to the elimination of the reservoirs and infected cells 18, especially having in mind that CCR5 Δ32/Δ32 homozygous does not mean a complete resistance to the HIV infection, due to the presence of CXC4-tropic HIV-1 or dual-tropic. In case of a positive selection of this variant, which normally shows a low frequency (in this patient it was 2.9%) 1,4,5, there are plenty of therapeutic approaches. For instance, the drug AMD3100 8,13 or a siRNA system such the one developed by RJ Pomerantz et al.9, which was quite remarkable. Nevertheless, the importance of this receptor in multiple events (immunomodulation, hematopoiesis, organogenesis and cerebellar neural migration) must be considered 9,10, because inhibition may mean a risk. For this reason, according to its low frequency, the complete reduction of its reservoirs and the stimulation of the Immune System to avoid its reseeding may turn out as a solution 1.

Also, the importance of the anti-HIV myeloablative therapy (although the low success rate raised by I. Huzicka F.18) was shown in two cases in which the DNA/RNA viral detection was negative. It was co-related to an intense pre-transplant cytoablative conditioning (Holland et al. & Contu et al.). The fact of reconstituting the immune system means an opportunity to generate a powerful response to an exhaustive viral burden, similar to the acute infection context 11,12,18. For this reason I think it may be interesting to isolate, stimulate and activate part of the innate immune component (IFN and NK cells) and T cytotoxic of the patient, in order to enhance the innate control events of viremia described in the next image 11,12.

 

Gene therapy for CCR5 Δ32/Δ32 phenotype generation:

June et al., by using engineered zinc-finger nucleases to disrupt endogenous CCR5 co-receptor in NOG mouse model, generated a robust and stable HIV-resistance genotype, both in vitro and in vivo, which increased the TCD4+ cells recount and reduced viremia (8300 copies/ml) compared with the control (60100 copies/ml). This technology can be designed to make a single break at a specific site in double-stranded genomic DNA, permanently modifying the genome via an imperfect repair by nonhomologous end joining (NHEJ). In this work mice were engrafted with ZFN-modified T CD4+ and showed the previously described results. This technology was also used on human primary T CD4+ cells, obtaining an efficacy and tolerability profile. For this reason an interesting clinic application is proposed 3.

In this context, it may be interesting to consider this technology for autologous CD34+ cells, in order to reconstitute an immune system with phenotype similar to the CCR5 Δ32/Δ32, via autologous stem cells. T cells transfection was accomplished by an adenovirus vector (Ad5/35) that encodes CCR5 ZFNs 3.

Naldini et al. tested the effectivity of the integrase defective lentiviral vectors to allow site specific gen addition through a process which required ZFN break and an homologous DNA template. This approach was carried out, for instance, in CD34+ cells incorporating a GFP cassette at the CCR5 site. Two different experiments showed an addition of the GFP cassete into the CCR5 locus in 80%-90% of the colony-forming cells analyzed 14. Therefore, another possible approach would be the insertion of a transgene in the CCR5 locus, to deplete its expression and thus generate a resistance phenotype to CCR5-tropic HIV-1.

One of the greatest advantages of this system is that it modifies the genotype of the cell without the need of integrating an alien DNA inside the genome since the expression systems used in this work  transiently expressed CCR5 ZFN. According to this, the use of integrative viral vectors which are related to insertional mutagenesis, becomes unnecessary 14,16.

Quoting Naldini et al.’s 14 paper: Gene correction overcomes the major limitations of gene- replacement strategies, as it restores both the function and expression control of the affected gene and avoids the risks associated with semi-random vector integration.

Aubourg et al. implemented a gene therapy in two patients with no matched bone marrow donors. It consisted in isolating CD34+ cells –from peripheral blood mononuclear cells stimulated with granulocyte colony-stimulating factor– by positive selection (with a immunomagnetic technique), and activate them with a mixture of citokines. Then, the cells got infected by replication-defective HIV-1–derived lentiviral vector (CG1711 hALD), which expressed a cDNA of the wild-type ABCD1 gene, which encodes the ALD protein. Transfected cells were then re-infused in both patients after a fully myeloablative conditioning regime with cyclophosphamide and busulfan. The process was clinically uneventful. Also, some months after both patients showed a clinical outcome improvement, similar to the one achieved by allogeneic HCT 2.

I believe that this therapy, through an ex-vivo CCR5 gene alteration, may be very interesting in order to reconstitute an hematopoietic system with a CCR5 Δ32/Δ32 phenotype. Nevertheless, while the aim in the X-linked Adrenoleukodystrophy case was the insertion of a wild-type gene 2, in the VIH resistance case it is necessary just the opposite thing. Hereby the combination of the successful Aubourg et al. approach 2 with the expression of siRNA system against CCR5 expression may turn out useful. Baltimore et al.  constructed a lentivirus-based vector to introduce siRNAs against CCR5 in peripheral blood T lymphocites. This system conferred an important protection against CCR5-tropic HIV-1 infection in these cells 15. However there are problems regarding the integrative viral vectors previously described, as the difficulties with large-scale manufacture in relation to the use of integrative viral vectors in clinical setting 6,17. Before this possible context, nonviral carriers which deliver native siRNA could also be considered, because they generate low immunogenicity and offer high structural and functional tunability 6.

I think all these approximations should be considered, as the continuous progress in correction and gene replacement system, in order to achieve the generation of a HIV resistance phenotype through an autologous HCT. The valuation of the advantages and disadvantages of different systems is necessary though, and particularly its long-term efficacy. For this reason it is also important employing a mouse model with a functional human immune system and an optimal HIV-1 infection profile. Since the major focus of Thiel at al.’ therapy is immune system reconstitution with HIV resistant phenotype, a mouse model which could accept efficient human HSC engraftment should be used. There is a model, the Rag2-/-gammaC-/- double knockout (DKO) mouse, which lacks T, B lymphocytes and NK cells, and in which the injection of CD34+ human HSC directly into the liver of new born DKO mice allows an efficient engraftment, and a reconstitution of the mouse with a functional human immune system in central and peripheral lymphoid organs 7. Using it may be interesting to value the reconstitution with a modified human HSC that expresses CCR5 Δ32/Δ32 phenotype.

Despite advances in gene therapy, this approach is very complicated and therefore in HIV infection would not be a feasible alternative to HAART, actually. However, I consider it an option to develop, especially in cases which HAART is not effective.

References:

  1. Gero Hütter, Daniel Nowak, Maximilian Mossner, Susanne Ganepola, Arne Musig, Kristina Allers, Thomas Schneider, Jorg Hofmann, Claudia Kucherer, Olga Blau, Igor W. Blau, Wolf K. Hofmann, Eckhard Thiel. Long-Term Control of HIV by CCR5 Delta32/Delta32 Stem-Cell Transplantation. New England Journal of Medicine. 2009; 360:692-8.
  2. Cartier N, Hacein-Bey-Abina S, Bartholomae CC, Veres G, Schmidt M, Kutschera I, Vidaud M, Abel U, Dal-Cortivo L, Caccavelli L, Mahlaoui N, Kiermer V, Mittelstaedt D, Bellesme C, Lahlou N, Lefrère F, Blanche S, Audit M, Payen E, Leboulch P, l’Homme B, Bougnères P, Von Kalle C, Fischer A, Cavazzana-Calvo M, Aubourg P. Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science. 2009; 326(5954):818-23.
  3. Perez EE, Wang J, Miller JC, Jouvenot Y, Kim KA, Liu O, Wang N, Lee G, Bartsevich VV, Lee YL, Guschin DY, Rupniewski I, Waite AJ, Carpenito C, Carroll RG, Orange JS, Urnov FD, Rebar EJ, Ando D, Gregory PD, Riley JL, Holmes MC, June CH. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nature Biotechnology. 2008; 26(7):808-16.
  4. Allers K, Hütter G, Hofmann J, Loddenkemper C, Rieger K, Thiel E, Schneider T. Evidence for the cure of HIV infection by CCR5{Delta}32/{Delta}32 stem cell transplantation. Blood. 2010.
  5. Hütter G, Thiel E. Allogeneic transplantation of CCR5-deficient progenitor cells in a patient with HIV infection: an update after 3 years and the search for patient no. 2. AIDS. 2011; 25(2):273-4.
  6. Shim MS, Kwon YJ. Efficient and targeted delivery of siRNA in vivo. FEBS J.2010; 277(23):4814-27.
  7. Zhang L, Meissner E, Chen J, Su L. Current humanized mouse models for studying human immunology and HIV-1 immuno-pathogenesis. Sci China Life Sci. 2010; 53(2):195-203.
  8. Kuritzkes DR. HIV-1 entry inhibitors: an overview. Current Opinion in HIV and AIDS. 2009; 4(2):82-87.
  9. Zhou N, Fang J, Mukhtar M, Acheampong E, Pomerantz RJ. Inhibition of HIV-1 fusion with small interfering RNAs targeting the chemokine coreceptor CXCR4. Gene Therapy. 2004; 11(23):1703-12.
  10. Mahalingam S, Karupiah G. Chemokines and chemokine receptors in infectious diseases. Immunology and Cell Biology. 1999; 77(6):469-75.
  11. Chang JJ, Altfeld M. Innate immune activation in primary HIV-1 infection. J InfectDis. 2010; 202 Suppl 2:S297-301.
  12. Chakrabarti LA, Simon V. Immune mechanisms of HIV control. Curr Opin Immunol. 2010;22(4):488-96.
  13. Hatse S, Princen K, Bridger G, De Clercq E, Schols D. Chemokine receptor inhibition by AMD3100 is strictly confined to CXCR4.FEBS Lett. 2002; 527(1-3):255-62.
  14. Lombardo A, Genovese P, Beausejour CM, Colleoni S, Lee Y, Kim KA, Ando D, Urnov FD, Galli C, Gregory PD, Holmes MC, Naldini L. Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat Biotechnol. 2007 Nov;25(11):1298-306. Epub 2007 Oct 28.
  15. Xiao-Feng Qin, Dong Sung An, Irvin S. Y. Chen, David Baltimore. Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5. PNAS. 2003 Jan; 7(100): 183-88.
  16. Escors D, Breckpot K. Lentiviral Vectors in Gene Therapy: Their Current Status and Future Potential. Arch. Immunol. Ther. Exp. (2010) 58:107–119.
  17. Couto LB, High KA. Viral vector-mediated RNA interference. Current Opinion in Pharmacology 2010, 10:534–542.
  18. I. Huzicka. Could bone marrow transplantation cure AIDS?: review. Medical Hypotheses 1999; 52(3): 247–257.
  19. Rich RR, Fleisher TA, Shearer WT, Schroeder HW, Frew AJ, Weyand CM. Clinical Immunology. Principles and Practices. Third edition. Mosby Elsevier; 2008.
Categorías:Inmunología
  1. 19 febrero, 2013 en 03:12

    Great article.

  2. 1 marzo, 2013 en 02:02

    What’s up, just wanted to mention, I liked this post. It was helpful. Keep on posting!

  3. 3 marzo, 2013 en 18:10

    My coder is trying to convince me to move to .
    net from PHP. I have always disliked the idea because of
    the expenses. But he’s tryiong none the less. I’ve been using WordPress
    on various websites for about a year and am concerned
    about switching to another platform. I have heard fantastic things about blogengine.
    net. Is there a way I can transfer all my wordpress posts into it?

    Any help would be really appreciated!

  4. 3 marzo, 2013 en 21:23

    WOW just what I was searching for. Came here by searching for nothing else but

  5. 4 marzo, 2013 en 06:18

    Hey There. I found your blog using msn. This is an extremely well written article.
    I will make sure to bookmark it and come back to read more of your useful information.
    Thanks for the post. I will definitely comeback.

  6. 4 marzo, 2013 en 19:03

    I’m more than happy to find this site. I wanted to thank you for ones time just for this fantastic read!! I definitely appreciated every little bit of it and i also have you bookmarked to check out new information in your site.

  7. 7 marzo, 2013 en 10:35

    I like it when people get together and share opinions.
    Great site, keep it up!

  8. 7 marzo, 2013 en 13:32

    That’s why you will benefit tremendously! Those lists can’t be known
    for some long Home Runs, too. As often as possible.

  9. 13 marzo, 2013 en 13:25

    Ahaa, its fastidious discussion concerning this piece of writing
    here at this website, I have read all that, so now
    me also commenting at this place.

  10. 15 marzo, 2013 en 08:55

    Hurrah! After all I got a weblog from where I be capable of actually take useful information regarding my study
    and knowledge.

  11. 4 abril, 2013 en 07:21

    Hey there, I think your site might be having browser compatibility issues.
    When I look at your blog site in Opera, it looks fine but when opening in Internet Explorer,
    it has some overlapping. I just wanted to give
    you a quick heads up! Other then that, terrific blog!

  12. 8 abril, 2013 en 07:35

    Hello, after reading this remarkable content i am as well glad to share my
    knowledge here with mates.

  13. 8 abril, 2013 en 11:51

    Hello, just wanted to tell you, I enjoyed this post.
    It was helpful. Keep on posting!

  14. 8 abril, 2013 en 12:09

    I really like your blog.. very nice colors & theme.
    Did you make this website yourself or did you hire someone to do it for
    you? Plz answer back as I’m looking to create my own blog and would like to know where u got this from. many thanks

  15. 8 abril, 2013 en 13:43

    In fact no matter if someone doesn’t be aware of after that its up to other users that they will assist, so here it takes place.

  16. 12 abril, 2013 en 08:16

    I’m gone to say to my little brother, that he should also visit this weblog on regular basis to get updated from most up-to-date news.

  17. 15 abril, 2013 en 16:13

    Hello, every time i used to check webpage posts here early in the daylight, since i love to learn
    more and more.

  18. 16 abril, 2013 en 03:42

    Excellent post! We are linking to this particularly great content on our website.
    Keep up the great writing.

  19. 17 abril, 2013 en 01:00

    Hi there, yup this editorial is really core and I have learned lot of
    things from it concerning blogging. thanks.

  20. 17 abril, 2013 en 07:50

    My spouse and i got now relieved that Michael could do his survey out of the precious recommendations he got out of
    the web page. It’s not at all simplistic just to always be giving away techniques which often others might have been trying to sell. And we also recognize we have the blog owner to give thanks to because of that. Most of the explanations you’ve made, the simple blog navigation, the
    relationships you make it possible to create – it’s got mostly remarkable, and it’s really facilitating
    our son and the family reckon that the theme is entertaining, and
    that is really fundamental. Thanks for all!

  21. 17 abril, 2013 en 09:41

    It’s difficult to find educated people in this particular subject, but you sound like you know what you’re talking about!
    Thanks

  22. 18 abril, 2013 en 01:54

    I visited several web sites except the value of current content at
    this website is truly wonderful.

  23. 18 abril, 2013 en 09:59

    We’re a group of volunteers and starting a new scheme in our community. Your web site offered us with valuable information to work on. You’ve performed
    an impressive task and our entire group will be grateful
    to you.

  24. 19 abril, 2013 en 01:52

    Please let me know if you’re looking for a writer for your blog. You have some really great posts and I think I would be a good asset. If you ever want to take some of the load off, I’d really like to write some material for your blog in
    exchange for a link back to mine. Please send me an email if interested.
    Many thanks!

  25. 19 abril, 2013 en 20:38

    I’m amazed, I have to admit. Rarely do I come across a blog that’s equally
    educative and engaging, and without a doubt, you have hit the nail on the head.

    The problem is something which too few folks are speaking intelligently about.

    I’m very happy that I stumbled across this during my hunt for something relating to this.

  26. 20 abril, 2013 en 13:27

    Great post. I was continuously checking this web-site and I am impressed!
    Very useful information specifically the closing section :)
    I deal with such information much. I used to be seeking this particular info for
    a long time. Thank you and best of luck.

  27. 20 abril, 2013 en 13:37

    Because the admin of this site is working, no uncertainty very quickly it will be famous, due to its quality contents.

  28. 21 abril, 2013 en 14:48

    Hello i am kavin, its my first occasion to commenting anywhere,
    when i read this post i thought i could also make comment due to this good article.

  29. 22 abril, 2013 en 09:12

    Hurrah, that’s what I was seeking for, what a data! present here at this web site, thanks admin of this web site.

  30. 23 abril, 2013 en 15:45

    Hi colleagues, its impressive entry about cultureand fully defined, keep it up all the time.

  31. 23 abril, 2013 en 16:56

    Having read this I believed it was very informative.
    I appreciate you finding the time and energy to put this informative article together.
    I once again find myself spending way too much time both reading and leaving comments.
    But so what, it was still worth it!

  32. 24 abril, 2013 en 03:06

    Hi! I know this is kind of off topic but I
    was wondering which blog platform are you using for this website?
    I’m getting fed up of WordPress because I’ve had issues with hackers and I’m looking at alternatives for another platform. I would be great if you could point me in the direction of a good platform.

  33. 27 abril, 2013 en 01:59

    Thanks in favour of sharing such a essential thinking, post is
    core, thats why i have read it completely

  34. 27 abril, 2013 en 12:20

    What’s Going down i’m new to this, I stumbled upon this I’ve discovered It absolutely helpful and it has helped me out loads. I am hoping to give a contribution & help other customers like its aided me. Great job.

  35. 28 abril, 2013 en 10:03

    Wow, incredible blog layout! How lengthy have you been blogging for?
    you make running a blog glance easy. The whole look of
    your site is wonderful, let alone the content material!

  36. 28 abril, 2013 en 11:45

    I visited various web pages but the class of current content
    at this web page is actually fabulous.

  37. 29 abril, 2013 en 20:17

    Omega-3 deficiency can also be linked to poor healing of wounds, flaking
    skin, dandruff and excema. Has your neck firming cream failed
    you and you now have resorted to wearing clothes with big collars or turtlenecks to hide unsightly neck wrinkles.
    With them, you can reap the full benefits of almond oil without any adverse effects.

  38. 30 abril, 2013 en 13:11

    Hence People with various skin problems can use almond oil products without
    having to worry about its adverse side effects. Finding the
    right hair conditioner can become costly, but now you can
    make your personalized conditioner at home for a fraction of the cost.
    If you are suffering from thin and brittle hair, almond oil can make your hair grow thicker and stronger over
    time as well.

  39. 30 abril, 2013 en 20:43

    It’s perfect time to make some plans for the longer term and it’s time to be happy.
    I’ve learn this post and if I may I desire to suggest you some attention-grabbing things or tips. Maybe you can write next articles regarding this article. I wish to learn even more issues about it!

  40. 1 mayo, 2013 en 21:21

    Now then, this weekend is nice in support of me, for the reason that
    this time i am reading this wonderful educational content here at my home.

  41. 2 mayo, 2013 en 15:13

    wonderful issues altogether, you simply won a new reader.
    What could you suggest about your post that you simply made some days ago?
    Any positive?

  42. 3 mayo, 2013 en 01:39

    Excellent article. Keep writing such kind of info on your site.
    Im really impressed by it.
    Hi there, You’ve performed a great job. I will definitely digg it and individually suggest to my friends. I’m sure they will be benefited
    from this site.

  43. 5 mayo, 2013 en 18:39

    Hello, I think your website might be having internet browser compatibility issues.
    Whenever I take a look at your blog in Safari, it looks
    fine but when opening in I.E., it’s got some overlapping issues. I simply wanted to give you a quick heads up! Besides that, fantastic website!

  44. 7 mayo, 2013 en 04:06

    Very good blog post. I absolutely love this site. Continue the good work!

  45. 7 mayo, 2013 en 10:09

    bbq time / beer time is approaching. If I can just go through this post in the next 10 minutes I’ll be able to unwind and have fun.

  46. 7 mayo, 2013 en 12:51

    If some one wishes to be updated with most recent technologies after that he must be visit this site
    and be up to date every day.

  47. 7 mayo, 2013 en 18:45

    Hello! Do you know if they make any plugins to protect against hackers?
    I’m kinda paranoid about losing everything I’ve worked hard on.

    Any recommendations?

  48. 8 mayo, 2013 en 08:38

    I do believe all of the concepts you have presented in your post.
    They are really convincing and can certainly work. Still, the
    posts are too brief for beginners. May just you please lengthen them a bit from next time?
    Thank you for the post.

  49. 8 mayo, 2013 en 09:21

    Hello There. I found your blog the use of msn.
    This is an extremely neatly written article.
    I will be sure to bookmark it and come back to read extra of your helpful info.
    Thanks for the post. I’ll certainly comeback.

  50. 8 mayo, 2013 en 15:39

    There is certainly a great deal to find out about this subject.
    I really like all the points you made.

  51. 8 mayo, 2013 en 19:06

    Hi, I do think this is an excellent blog. I stumbledupon it ;
    ) I’m going to return once again since i have saved as a favorite it. Money and freedom is the best way to change, may you be rich and continue to guide others.

  52. 11 mayo, 2013 en 09:01

    Excellent blog here! Also your website loads up very fast!
    What web host are you using? Can I get your affiliate link to your host?
    I wish my web site loaded up as quickly as
    yours lol

  53. 12 mayo, 2013 en 04:22

    This text is priceless. Where can I find out more?

  54. new
    13 mayo, 2013 en 19:20

    Because the admin of this website is working, no doubt very soon it will be well-known, due to its quality
    contents.

  55. 14 mayo, 2013 en 17:49

    Hi there. I’m sorry to bother you but I ran across your blog and noticed you happen to be using the exact same template as me. The only problem is on my site, I’m unable to get the layout looking
    like yours. Would you mind emailing me at: jermainehefner@live.
    de so I can get this figured out. By the way I’ve bookmarked your web-site: http://bioxano.wordpress.com/2011/04/21/%E2%80%9Cberlin-patient%E2%80%9D-and-gene-therapy-against-hiv-infection/ and will be visiting often. Thanks!

  56. 16 mayo, 2013 en 13:06

    Write more, thats all I have to say. Literally, it seems as though you relied on the video to make your point.
    You obviously know what youre talking about, why waste your intelligence on just posting videos to your site when you could be giving us something enlightening to read?

  57. 16 mayo, 2013 en 16:14

    Thanks for any other informative site. The place else could I get that kind of info written in such a perfect approach?

    I’ve a project that I’m just now working on, and I have been on
    the glance out for such info.

  58. 16 mayo, 2013 en 20:32

    My wife and I stumbled over here coming from a other website and assumed I might as well check things out.
    I like things i see so now i am following you.
    Look forward to going over your web page again.

  59. 17 mayo, 2013 en 04:18

    Hiya! I know this is kinda off topic but I’d figured I’d ask.
    Would you be interested in trading links or maybe guest writing a blog post or
    vice-versa? My website covers a lot of the same subjects as yours and I feel
    we could greatly benefit from each other. If you happen to be interested feel free to
    send me an e-mail. I look forward to hearing from you!
    Great blog by the way!

  60. 17 mayo, 2013 en 07:12

    I do not know whether it’s just me or if perhaps everybody else experiencing problems with your blog. It looks like some of the text in your posts are running off the screen. Can someone else please provide feedback and let me know if this is happening to them too? This might be a problem with my browser because I’ve had this happen previously.
    Thanks

  61. 18 mayo, 2013 en 08:04

    Hi! I’m on the brink of start a new site. I’m sort of familiar by using Dreamweaver, but that’s about it. Will that be sufficient, or must I also need to know HTML as well as Cascading stylesheet? I wouldn’t like to make things too tricky for myself.
    So if everything else stops, I may just go with a web theme and work from that point.
    Any suggestion would be very valued.

  62. 18 mayo, 2013 en 10:45

    Hi, i read your blog occasionally and i own a similar one and i
    was just curious if you get a lot of spam remarks?
    If so how do you reduce it, any plugin or anything you can suggest?
    I get so much lately it’s driving me insane so any help is very much appreciated.

  63. 18 mayo, 2013 en 13:14

    Do you have a spam problem on this site; I also am a blogger, and
    I was wanting to know your situation; many of us have
    developed some nice practices and we are looking to exchange solutions with others, be sure to shoot me an
    email if interested.

  64. 18 mayo, 2013 en 15:18

    Write more, thats all I have to say. Literally, it
    seems as though you relied on the video to make your point.
    You clearly know what youre talking about, why waste your intelligence on just
    posting videos to your weblog when you could be giving us something enlightening to read?

  65. 18 mayo, 2013 en 21:25

    I’m really impressed with your writing skills and also with the layout on your weblog. Is this a paid theme or did you modify it yourself? Anyway keep up the nice quality writing, it’s
    rare to see a nice blog like this one today.

  66. 19 mayo, 2013 en 06:50

    Hey! This is my first visit to your blog! We are a collection of volunteers and starting a new initiative in a community
    in the same niche. Your blog provided us useful information to work on.
    You have done a extraordinary job!

  67. 19 mayo, 2013 en 11:17

    I am new to developing sites and I was wanting to know if having your website title related to your articles and other
    content really that important? I notice your title, ““Berlin Patient” and gene therapy against HIV infection | Bio-Xano ” does seem to be spot on with what your website is about yet,
    I prefer to keep my title less content descriptive and based more around site branding.
    Would you think this is a good idea or bad idea? Any kind of assistance would be greatly appreciated.

  68. 19 mayo, 2013 en 11:26

    I own a new pilates studio in the North. Believe that or not, I don’t possess a webpage at this point. The issue is I am not sure whether to use Blogger or Hubpages. I discover that I want a lot more of a weblog feel than a webpage feel, if that makes any sense. I need to have the ability to update my website with weblog posts. Would you be capable of give a couple of positives and negatives for going with either Blogger or Live journal? I definitely want to come to a choice relatively swiftly on this , since it does not make sense for me to own this business without having a site. It would be kind if you could give me some assistance. Please write to: dongdonohue@live.com if you have some spare time. Thanks, and btw very good site, i wish my blog to be like the one you have one day!

  69. 20 mayo, 2013 en 06:19

    Hey there! This is my first comment here so I just wanted
    to give a quick shout out and tell you I genuinely enjoy reading your articles.
    Can you suggest any other blogs/websites/forums that cover the same topics?
    Thanks a ton!

  70. 20 mayo, 2013 en 10:59

    Interesting blog! Is your theme custom made or did you download it from somewhere?
    A design like yours with a few simple adjustements would really make my blog shine.
    Please let me know where you got your design. Cheers

  71. 20 mayo, 2013 en 13:34

    cash advances Research For Posts In Ideamarketers Short Article
    Listing
    Wise credit rating card people know how to get totally free use of
    anyone else’s funds every single month, by making use of their credit score card and then shelling out the full stability right before finance costs are assessed.
    Credit card is an important economical instrument these times as it extends line of credit history to the specific. Even some credit history cards provide cash withdrawal up to selected limitations as well. Credit rating card features lot of advantage to an particular person as an specific will not have to hold hard cash with him and it also minimizes possibility of losing dollars as perfectly.
    The truth is that bad credit score ratings do not necessarily mean it is the stop of the highway for individuals who are in need to have of funding, both for modest individual loans or entire home finance loan loans. Approval is however attainable, in particular if sure features are tackled.
    A variety of United kingdom Embassies throughout the world are urging the nations that they characterize to use now for their Uk visa if they are hoping to make it to the London 2012 Olympic and Paralympic game titles. In a bid to deal with the envisioned influx of individuals set to arrive on British shores this summer time, British isles Immigration has presently started processing British isles Visa applications to fulfill the increasing need.
    The better credit history boundaries possibly will never current a challenge for those who are cautious about how they use credit. Obtaining additional credit score out there doesn’t mean you have to use it,
    and fiscally dependable individuals will command their paying, no
    subject what their credit history limitations are. These customers
    can take pleasure in the privelege of a higher credit rating score, and the better funding offers that go with it.

    Following are some guidelines on how to begin establishing a superior cash advances record.
    There is no particular get that you will need to adhere to.
    Nonetheless, we strongly recommend that you try out to locate out about your recent credit circumstance.
    Initial of all, acquire a cost-free duplicate of your credit history report to see if you now have some credit rating history and you failed to
    know it.
    And hold in intellect your credit rating utilization ratio is not a fastened selection it can adjust substantially above the class
    of one particular month, relying on when you pay out your monthly bill and
    when the creditor reports your payment and harmony to the credit score bureau.

    Apart from this, because of to the reality just
    one unique can use the cell phone only until these time that it has
    credit rating on it, one can not excessively use the mobile cell phone
    and induce one’s fees to go up exponentially. cash advances

  72. 20 mayo, 2013 en 18:10

    It is really strange, i was on your site just 15 minutes ago, when my browser was freezing, did you added some new plugins or anything else which could cause such a behaviour?

    I visit your site pretty regulary and never had that before!

  73. 21 mayo, 2013 en 01:25

    Hello just wanted to give you a quick heads up. The words in your article seem to be running
    off the screen in Internet explorer. I’m not sure if this is a formatting issue or something to do with browser compatibility but I thought I’d post to let you
    know. The design and style look great though! Hope you get the problem fixed
    soon. Many thanks

  74. 21 mayo, 2013 en 18:01

    I know this site gives quality dependent posts and additional information,
    is there any other site which presents such data in quality?

  75. 23 mayo, 2013 en 19:42

    I do believe all the points you have presented for your post.
    They’re extremely convincing and can certainly work.
    Still, the posts are quite quick for beginners. Many thanks for the post.

  76. 24 mayo, 2013 en 16:49

    Howdy, i ‘m a blogger like you, and was thinking if you by chance experimented with those addthis.com follow this buttons and if they will be any good. I recognized they will load a ton of code, so i am uncertain if its well worth adding them, due the quite a lot increase of KB of the total filesize. Have you ever tried it? I am excited to hear what other people say about that? Please sent me a message to maddison.castellano@bigstring.com if you have any experience with those add-ons. Thx .. btw, luv the blog ever since!

  77. 25 mayo, 2013 en 02:39

    Art & Fine can be a terrific website ….

  78. 25 mayo, 2013 en 08:07

    Wow, this article is good, my younger sister is analyzing such
    things, so I am going to convey her.

  79. 25 mayo, 2013 en 14:40

    Excellent post. Keep writing such kind of
    information on your site. Im really impressed
    by it.
    Hey there, You have done a fantastic job. I will certainly
    digg it and personally suggest to my friends.
    I’m sure they will be benefited from this website.

  80. 26 mayo, 2013 en 03:14

    As the admin of this web site is working, no question very quickly it will be
    well-known, due to its quality contents.

  81. 29 mayo, 2013 en 13:09

    I admire your work, thank you for sharing all the beneficial blog posts.

  82. 29 mayo, 2013 en 15:59

    I believe this website got some truly useful stuff on it!

  83. 30 mayo, 2013 en 07:50

    I am now not positive the place you are getting your
    info, but great topic. I must spend some time studying more or working out more.
    Thank you for magnificent info I used to be searching for this information
    for my mission.

  84. 3 junio, 2013 en 17:39

    Thanks a lot for sharing this with all people you actually know what you’re speaking about! Bookmarked. Kindly additionally discuss with my website =). We will have a hyperlink alternate contract between us

  85. 4 junio, 2013 en 01:59

    Wow that was odd. I just wrote an incredibly long comment but after I clicked submit my comment didn’t show up. Grrrr… well I’m not writing all
    that over again. Anyhow, just wanted to say superb blog!

  86. 4 junio, 2013 en 02:29

    Your means of telling the whole thing in this piece of writing is
    genuinely good, all be able to easily know it, Thanks a lot.

  87. 4 junio, 2013 en 02:52

    Hi, Neat post. There is an issue with your web site in internet explorer,
    may check this? IE still is the market chief and a huge component of people will miss your magnificent writing because of this problem.

  88. 4 junio, 2013 en 04:16

    Hi, I log on to your blogs on a regular basis. Your humoristic style is awesome, keep up the good work!

  89. 5 junio, 2013 en 08:24

    You could certainly see your expertise within the article
    you write. The world hopes for even more passionate writers
    such as you who aren’t afraid to say how they believe. Always follow your heart.

  90. 6 junio, 2013 en 04:36

    I all the time used to read paragraph in news papers but now as I am a user of internet therefore from now I am using net for content,
    thanks to web.

  91. 6 junio, 2013 en 13:37

    Every weekend i used to pay a visit this web page, because i wish for enjoyment,
    since this this web page conations really nice funny
    material too.

  92. 6 junio, 2013 en 19:27

    My programmer is trying to convince me to move to .
    net from PHP. I have always disliked the idea because of the expenses.
    But he’s tryiong none the less. I’ve been using WordPress on
    a number of websites for about a year and am nervous about switching to another platform.

    I have heard great things about blogengine.
    net. Is there a way I can transfer all my wordpress posts
    into it? Any kind of help would be greatly appreciated!

  93. can i buy viagra in canada
    7 junio, 2013 en 13:06

    The more you push yourself too far, the less motivated
    you will be to continue with your program, and the more you put your life in
    danger. More than a decade later, Viagra remains to be
    one of the most popular impotence treatments for men.

    Men who have been suffering from erectile problems for a prolonged
    period of time can treat them with the help of prescription medications such as Viagra.

  94. 8 junio, 2013 en 08:09

    Your post features established necessary to me. It’s quite educational and you’re simply obviously very knowledgeable in this area. You get popped my own sight in order to varying views on this matter with intriguing, notable and sound content.

  95. 10 junio, 2013 en 06:24

    Hello to every one, the contents present at this website are in fact awesome for people experience, well, keep up the good work fellows.

  96. 10 junio, 2013 en 20:15

    Great article, i surely love this web site, keep it up.

  97. how to buy valacyclovir online uk
    11 junio, 2013 en 09:58

    Performance anxiety is the commonest cause of erectile dysfunction that there is.
    It is often referred to as impotence, which actually includes all of the above mentioned sexual problems along with erectile
    problems. The majority of available penis pills are made from a blend of natural ingredients, herbs,
    vitamins and herbal supplements that stimulate the
    growth of safe and effective.

  98. 12 junio, 2013 en 19:27

    rgndxdwchsly

  99. 12 junio, 2013 en 19:28

    uyzztomgqfgc

  100. 14 junio, 2013 en 11:57

    If you want to improve your experience simply keep visiting this site and be updated with the most recent gossip posted here.

  101. 16 junio, 2013 en 19:15

    The three main types of retaining walls are gravity walls, piling walls,
    anchored walls or cantilever walls. This happens because the landscape design does not have balance and
    symmetry. You will find information there on a lot of topics, including sustainable landscape design
    , designing an English country garden, costings and much more.

  102. 17 junio, 2013 en 09:40

    Nice blog here! Also your website loads up very fast!
    What host are you using? Can I get your affiliate link to your host?
    I wish my website loaded up as fast as yours lol

  103. 17 junio, 2013 en 18:11

    My family every time say that I am killing
    my time here at net, except I know I am getting experience daily by reading
    thes nice articles or reviews.

  104. 18 junio, 2013 en 07:26

    I love it when people get together and share thoughts.
    Great website, continue the good work!

  105. 18 junio, 2013 en 09:08

    Good day! This is my first comment here so I just wanted to give a quick shout out and say I really enjoy reading through your blog posts.
    Can you recommend any other blogs/websites/forums that go over the same topics?
    Thanks for your time!

  106. 19 junio, 2013 en 00:56

    Training English In Greece
    These days, although the English Shepherd has attained an underground recognition as adaptable working and companion
    puppy, the breed stays alternatively exceptional exterior of its native United States.

    Bulldogs are people pleasers and want to please their family members.
    This tends to make English bulldogs easy to practice with suitable procedures.
    They call for day by day treatment and attention, but are not extremely
    demanding.
    Since we are likely by means of noticeable adjustments in lending currently, it is incredibly vital you know where you now stand with your credit history rating.
    There are some variations heading on in FHA as properly, even although FHA does not have a credit rating score requirement the bank that purchases these loans may.
    So if you have reduced credit scores, you could want to maximize that rating even to get a FHA loan in this present-day industry.
    Get a current duplicate of your free of charge credit score score report, and if you have concerns there has never been a much better time
    than now to perform on your credit rating so you
    can purchase in the in close proximity to foreseeable future.

    You can Google-up a lot of handy resources on this subject matter, that will assist you
    fortify your information and make creating for the target viewers diverse than your very own substantially simpler and additional pleasurable.

    Whilst text loans and Welsh parish documents extend back as
    considerably as 1538, not all will have survived the ravages
    of hearth and flood, so you should not expect to be ready to sail back as far as this day!
    The previously records have been recorded on paper, but from 1558 onwards the extra tough parchment (designed
    from sheepskin) was employed in desire. Even so, extremely few parish
    history endure just before the 1600s.
    Alternatively than this bizarre behaviour it truly is
    greater to give your baby a rationale to use English phrases and this can
    be carried out by way of language online games, plays and skits, tales and tunes.
    Now with young ones below 3 you do not use structured
    language games but relatively enjoy along with the boy or girl, chatting away employing simple
    language and repeating the precise identical words normally.
    You are relying on the point that the subconscious
    will possibly be picking up the seems and coming into the databases, and rest confident they will be!

    There are a lot of ways to approach the challenge of university
    student attitudes. We attempt to support college
    students turn into a lot more successful learners though operating in a supportive setting, by way of particular evaluation and responses, via
    use of bodily or aggressive jobs and layered action studying.
    We also believe a social community of instructors and pupils is handy when you face
    mastering barriers. There are a lot of procedures but only one particular target.

    Even however it is a fairly younger place in
    mild of planet background, the United States
    has turn out to be a world superpower, exclusively just
    after Entire world War II. The united states has a hand in politics, economics, and culture about the entire world.
    As such, English is getting to be more and more important as a global language.
    When you include the impact of The us in today’s worldwide financial system to the historic and present-day impact of Wonderful Britain, knowledge English gets nearly critical to carrying out enterprise in the existing economic sector. payday loans online online loan

  107. 19 junio, 2013 en 08:40

    Hi mates, pleasant article and pleasant arguments commented at this place, I am
    actually enjoying by these.

  108. 22 junio, 2013 en 05:45

    Great post. I used to be checking continuously this blog
    and I am inspired! Very useful info specifically the closing section :
    ) I maintain such information much. I was seeking this certain information for a long time.
    Thank you and good luck.

  109. 22 junio, 2013 en 10:27

    vvbkkvpuvyuh

  110. 22 junio, 2013 en 16:51

    I think this is one of the most important info
    for me. And i am glad reading your article. But wanna remark on some general things, The site style is perfect, the articles is really great : D.
    Good job, cheers

  111. 22 junio, 2013 en 18:49

    Howdy are using WordPress for your site platform? I’m new to the blog world but I’m
    trying to get started and set up my own. Do you need any coding knowledge to make your own
    blog? Any help would be greatly appreciated!

  112. 25 junio, 2013 en 07:40

    hey there! , Everyone loves your own creating thus a whole lot! quantity we connect extra regarding your publish upon Google? We need a professional within this household to be able to uncover the challenge. May very well be that is anyone! Taking a look forward to check out anyone.

  113. 27 junio, 2013 en 01:33

    cnwskksmgmty

  114. 28 junio, 2013 en 06:03

    Asking questions are really pleasant thing if you are not understanding anything fully,
    but this piece of writing offers good understanding yet.

  115. 28 junio, 2013 en 21:25

    Excellent pieces. Keep writing such kind of information
    on your blog. Im really impressed by it.
    Hi there, You’ve done a great job. I’ll definitely digg it and for my part
    recommend to my friends. I’m sure they’ll be benefited from
    this website.

  116. 29 junio, 2013 en 06:25

    I loved as much as you will receive carried out right here.

    The sketch is attractive, your authored material stylish.
    nonetheless, you command get got an impatience over that you wish be
    delivering the following. unwell unquestionably come more formerly again since exactly
    the same nearly very often inside case you shield this increase.

  117. 2 julio, 2013 en 06:24

    ufinszmsmgjk

  118. 3 julio, 2013 en 14:35

    I am really impressed with your writing skills and also
    with the layout on your blog. Is this a paid theme or did you modify
    it yourself? Anyway keep up the excellent quality writing, it’s rare to see a nice blog like this one today.

  119. 4 julio, 2013 en 07:11

    Good way of explaining, and pleasant post to get information regarding my
    presentation subject, which i am going to present in school.

  120. 4 julio, 2013 en 20:36

    I constantly spent my half an hour to read this website’s posts daily along with a cup of coffee.

  121. 5 julio, 2013 en 07:13

    Great post.

  122. 7 julio, 2013 en 05:29

    I’m already busy watching Real Chance of Love, with Real and Chance attempting to girl swap every week, and frankly I think I’ve had just about enough of double duos trying to hook up with the same damn set of people.
    Half the population hadn’t heard of them till MTV decided to air them on Double Shot at Love. While she’s the youngest
    MILF of the bunch at 29, Alba has admitted that she believes “dieting is for the birds” and she’s “not a big workout person”.

  123. 7 julio, 2013 en 15:07

    What i don’t realize is actually how you are not actually much more neatly-favored than you may be now. You are so intelligent. You know therefore significantly with regards to this subject, made me personally believe it from numerous numerous angles. Its like women and men aren’t interested unless
    it is one thing to accomplish with Girl gaga! Your individual stuffs nice.
    All the time maintain it up!

  124. 8 julio, 2013 en 12:35

    Hi there to all, it’s in fact a pleasant for me to pay a visit this web site, it contains priceless Information.

  125. 9 julio, 2013 en 09:51

    Thank you, I’ve just been searching for info approximately this subject for a while and yours is the best I have discovered till now. But, what about the bottom line? Are you sure about the supply?

  126. 9 julio, 2013 en 19:08

    hello!,I love your writing very so much! percentage we keep
    in touch more approximately your article on AOL? I require
    a specialist in this space to resolve my problem. Maybe that’s you! Taking a look forward to peer you.

  127. 10 julio, 2013 en 07:07

    You are so awesome! I don’t believe I’ve truly read a single
    thing like that before. So wonderful to find someone with some original thoughts on
    this issue. Seriously.. thanks for starting this up. This website is one thing that is required on the internet,
    someone with a bit of originality!

  128. 10 julio, 2013 en 08:56

    I got this site from my friend who told me on the topic of
    this website and at the moment this time I am browsing this site and reading very informative articles
    or reviews at this place.

  129. 10 julio, 2013 en 18:43

    I am actually delighted to read this webpage posts which
    includes lots of helpful data, thanks for providing these kinds of information.

  130. 11 julio, 2013 en 00:29

    I am sure this piece of writing
    has touched all the internet visitors, its really really nice piece of writing on
    building up new web site.

  131. 30 agosto, 2013 en 07:15

    Hi, every time i used to check web site posts here early in the break of day, since i like to gain knowledge of more and more.

  132. 22 noviembre, 2013 en 11:49

    Hello” thankGod for the wonderful healing spell power which smart render to my life which oracle support, i was been diagnosed with HIV-AIDS over 7years now which has lead me to unployment and loosing my relationship with my fiance after he heard that i am suffering from the VIRUS, i tried begging him to allow me stays with him, but he still insist on letting me out of his home, until a friend of mine on facebook from SOUTH AFRICA told me more about smart the great healer, that he is going to help restore and healed my diseased with his powerful healing spell, she send me of smart email address and i quickly contacted him, and he replied to after 30mins of my message that my disease is a little and minor disease, that he will solved the disease immediately only if i can accept all he requested from, i told him yes, because truly i know that ” HIV-AIDS IS A WICKED AND DEADLY DISEASE I EVER KNOWS “. so i did all he want from me and suprisingly on friday last week smart called me that my problems is solvesd that i should hurry up to the hospital for a checkup, which i truly really did, i comfirm from my doctor that i am now HIV-NEGATIVE AND NOT LONG +, I jump up and was crying heavily because truly the disease drive alot of things away from my life, my friends, my cousin, my fiance and relatives, but now since i was comfirm NEGATIVE- my lost friends and fiance has been back to me begging for forgiveness, i have forgiven them and now we are now best of friend. again i will say to smart that he is blessed and may the almighty God which i serve raise him and his family to the higher level in life. Thank and god bless. Please email smart for healing and solution now at = destinysmart11@gmail.com
    FROM U.S.A

  133. Ada
    19 junio, 2014 en 01:56

    I blog often and I genuinely appreciate your content. The article has truly peaked my interest.I am going to take a note of your site and keep checking for new details about once per week. I opted in for your Feed as well.my blog :: click here

  134. 28 agosto, 2014 en 10:03

    Lenders also stress the simplicity of applying for financing for these trucks, and looking at lenders’ websites, the process does seem simple.Small business vehicle financing may be a viable option in such cases.my page – buy commrical trucks with lease

  135. 29 agosto, 2014 en 08:49

    Quality posts is the main to interest the users to visit the web site, that’s what this web page is providing.For the best solution please check out this post :: post workout formula

  1. 28 noviembre, 2013 en 09:23
  2. 17 marzo, 2014 en 07:33
  3. 20 marzo, 2014 en 02:11
  4. 20 marzo, 2014 en 02:11
  5. 22 marzo, 2014 en 03:26
  6. 24 marzo, 2014 en 02:24
  7. 26 marzo, 2014 en 04:39
  8. 27 marzo, 2014 en 08:19
  9. 29 marzo, 2014 en 02:15
  10. 29 marzo, 2014 en 04:36
  11. 1 abril, 2014 en 02:18
  12. 1 abril, 2014 en 02:22
  13. 1 abril, 2014 en 11:43
  14. 1 mayo, 2014 en 11:33
  15. 1 mayo, 2014 en 18:17
  16. 7 mayo, 2014 en 10:44
  17. 21 mayo, 2014 en 03:30
  18. 21 mayo, 2014 en 14:59
  19. 24 mayo, 2014 en 19:39
  20. 31 mayo, 2014 en 08:26
  21. 3 junio, 2014 en 07:59
  22. 4 junio, 2014 en 17:41
  23. 5 junio, 2014 en 14:37
  24. 6 junio, 2014 en 17:11
  25. 13 junio, 2014 en 17:09
  26. 18 junio, 2014 en 02:07
  27. 18 junio, 2014 en 16:32
  28. 10 julio, 2014 en 02:29
  29. 16 julio, 2014 en 04:33
  30. 16 julio, 2014 en 14:32
  31. 23 julio, 2014 en 19:40
  32. 24 julio, 2014 en 13:36
  33. 6 agosto, 2014 en 06:35
  34. 12 agosto, 2014 en 16:01
  35. 18 agosto, 2014 en 22:27
  36. 18 agosto, 2014 en 22:34
  37. 30 agosto, 2014 en 10:12
  38. 1 septiembre, 2014 en 11:24
  39. 3 septiembre, 2014 en 18:43
  40. 10 septiembre, 2014 en 19:02
  41. 11 septiembre, 2014 en 08:13
  42. 8 octubre, 2014 en 21:02
  43. 10 octubre, 2014 en 18:58
  44. 13 octubre, 2014 en 04:41
  45. 13 octubre, 2014 en 19:52
  46. 14 octubre, 2014 en 04:48
  47. 15 octubre, 2014 en 16:48
  48. 15 octubre, 2014 en 16:55
  49. 15 octubre, 2014 en 16:57
  50. 16 octubre, 2014 en 13:56
  51. 21 octubre, 2014 en 14:10
  52. 30 octubre, 2014 en 02:49

Deja un comentario

Introduce tus datos o haz clic en un icono para iniciar sesión:

Logo de WordPress.com

Estás comentando usando tu cuenta de WordPress.com. Cerrar sesión / Cambiar )

Imagen de Twitter

Estás comentando usando tu cuenta de Twitter. Cerrar sesión / Cambiar )

Foto de Facebook

Estás comentando usando tu cuenta de Facebook. Cerrar sesión / Cambiar )

Google+ photo

Estás comentando usando tu cuenta de Google+. Cerrar sesión / Cambiar )

Conectando a %s

Seguir

Recibe cada nueva publicación en tu buzón de correo electrónico.

A %d blogueros les gusta esto: