Future Application

“The whole is more than the sum of its parts.” The very same principle is valid for OPTOPTOSIS. Besides our idea to induce apoptosis with the help of optogenetics, we expanded our construct into a whole concept. We did not only build two “kill-switches”, we also thought about what would come next. This means we developed a strategy how to get our construct in cancer cells and found various solutions how to get the light to the target tissue.

Viral Vectors

Viral vectors are modified viruses that are used in medical or biological research to insert specific genes into a test-organism. The concept of viral vectors is based on the ability of viruses to inject their genome into a cell while being specific to the membrane proteins of the particular target cell.

The virus replication is inhibited due to the deletion of some of its genes, so the virus needs a "helper virus" to complete their viral life cycle. These viruses are part of new approaches in cancer treatment, but this technique still is in the developmental stage because of its side effects.

Gene therapy uses a lot of different viral vectors depending on the application. Commonly used viral vectors are Adenoviruses, AAVs (Adeno-associated viruses), Herpes simplex or Lentiviruses.

AAVs belong to the group of so called satellite viruses, which depend on the help of other, non-related viruses (Adenoviruses in this case) to be infectious. AAVs that enter a cell cause less to no damage to the organism and therefore are ideal for gene therapy, as they can transport genes to certain tissues without causing any disease in the host organism. ^[1]

Adenoviruses themselves are icosahedral double-strand DNA viruses, which cause harmless infections of the respiratory system in humans and are often used as vectors in gene therapy. ^[2]

Herpes simplex viruses derivatives or hybrids with suitable viruses are other viral-based delivery systems for gene therapy. The natural property of a long term infection without symptoms suits these viruses perfect for a stable transcription of a specific gene product. In example, for a therapy a HSV-1/AAV hybrid can be advantageous with the specificity of the AAV and the replication as well as the capacity of the HSV-1, making this hybrid perfect for the transduction of large DNA-fragments. ^{[3] [4] [5] [6] [7]}

Retroviruses can also be utilized as a viral-based vector systems. MLV (Moloney leukemia virus) and HIV-1(human immunodeficiency virus) are used in gene therapy. This retroviral approach is a longterm expression system due to the genome integration of the inserted genes into the host genome. ^[8] Lentiviruses have been already used as vectors in clinical trials and can potentially be a powerful tool in gene therapy. ^[9][10][11]

Our constructs could be inserted into the targeted cells with AAVs, adenoviruses or even with a HSV-1/AAV-like hybrid vector system. Once infected, these cells would be able to express the blue light switch and the red light switch. It would be necessary that two viruses infect the same cell to ensure interaction due to the size of our constructs. A double infection would only be necessary if AAVs or Adenoviruses alone would be used for transduction. This can be avoided if e.g. a AAV/HSV-1 Hybrid is used.

Non-viral approaches in gene therapy are also being developed, making use of the Crispr-cas9 editing system. Single gene-knockout or even knock-in can be used with this system making it suitable for gene therapy. The human immune system can be an obstacle in this therapeutical approach. ^[12]

Another non-viral approach would be the use of so called minicircle DNA, a vector system used for the transfection of mammalian cells. All prokaryotic sequences have been removed from the plasmid leading to a great size-reduction of the vector and therefore significantly increasing the transfection efficiency and expression rate compared to conventional plasmids. ^[13]

Viral vectors are a significant component in establishing our optogenetical kill-switches for future therapeutical usage, thus discussing the procedure of establishing gene therapeutical medicaments and ethical questions were of high importance to us.

The Light Into Tissue Struggle

To treat tumors effectively it is necessary for light to reach the affected organ with sufficient intensity. For PhytochromeB best results were achieved when it was exposed to over 60 nmol*cm-2. The optical density of live tissue is highly dependent on the length of the electromagnetic waves. At the required wavelength of 473 nm the blue light only reaches about 1 mm into tissue. At 660 nm however the absorption coefficient is lower, so red light can reach a depth of 1 cm. ^[14]

To overcome the hurdle of a high absorption coefficient we developed different approaches. UCNPs stands for Upconversion Nanoparticles, artificial molecules with the feature of changing the wavelength of light. When exposed to near infrared (NIR) light with a wavelength of 980 nm, UCNPs emit light of another wavelength, 475 nm for example. ^[15]

Near infrared light travels further into tissue than blue light. Using this effect UCNPs found use in photodynamic therapies to kill deep tumors as a result of the more effective penetration of NIR light. Gang Han PhD. kills tumors in a depth greater than 1 cm with the aid of the artificial molecules. ^[16]

No bigger than 70 nm, the UCNPs could either be injected in the bloodstream, which raises the question of how to remove them afterwards, or they could be attached to proteins. To fix UCNPs on proteins streptavidin is installed on the surface, which interacts with a StrepTag that is synthesized on proteins.^[17]

To expose tumors in depths NIR cannot reach, the use of optical fibers becomes inevitable. The fibers direct light of any color to their tip exploiting the effect of total reflection. Being less invasive than common surgeries in cancer treatment due to a thickness of a few µm is the significant advantage. Also, the minuscule diameter allows immense precision for the illumination.

Optical fibers are already used in mice to test the change of behavior when brain cells are exposed to light. This application, aiming to heal mental conditions like Parkinson’s Disease and Dementia is proof for the applicability of optical fibers in optogenetics and surgeries.

References

^[1]= Madigan et al., Brock Mikrobiologie (Pearson, 13th edition), p. 371
^[2]= Madigan et al., Brock Mikrobiologie (Pearson, 13th edition), p. 931
^[3]= Saydam et al.2015, construction and packaging of herpes simplex virus/adeno-associated virus (HSV/AAV) Hybrid amplicon vector https://www.ncbi.nlm.nih.gov/pubmed/22383640
^[4]= Andreas Jacobs et al. 1999,HSV-1-Based Vectors for Gene Therapy of Neurological Diseases and Brain Tumors: Part II. Vector Systems and Applications https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1508111/
^[5]= Marconi et al.2015, Herpes simplex virus type 1(HSV-1)-derived recombinant vectors for gene transfer and gene therapy https://www.ncbi.nlm.nih.gov/pubmed/25431072
^[6]= Published online 2013 Apr 23,https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3908892/figure/F1/
^[7]= Michele Simonato et al.2013 Apr 23,Progress in gene therapy for neurological disorders https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3908892/
^[8]= José Eduardo Vargas, Leonardo Chicaybam et al. 2016 Oct 12, Retroviral vectors and transposons for stable gene therapy: advances, current challenges and perspectives http://translational-medicine.biomedcentral.com/articles/10.1186/s12967-016-1047-x
^[9]=Seassa M. et al. 2016 Jul 30, Lentiviral haemopoietic stem-cell gene therapy in early-onset metachromatic leukodystrophy: an ad-hoc analysis for a non-randomised, open-label, phase 1/2 trial https://www.ncbi.nlm.nih.gov/pubmed/27289174
^[10]=Nasirinezhad et al. 2015 Jan 7 , Viral vectors encoding endmorphins and serine histogranin attenuate pain symptoms after spinal cord injury in rats https://www.ncbi.nlm.nih.gov/pubmed/25563474#
^[11]=Mautino, Morgan. 2002 Jan 16, Gene therapy of HIV-1 infection using lentiviral vectors expressing anti-HIV-1 genes https://www.ncbi.nlm.nih.gov/pubmed/11839215
^[12]=Fogleman et al. 2016 Aug 20, Crispr/cas9 and mitochondrial gene replacement therapy: promising techniques and ethical considerrations https://www.ncbi.nlm.nih.gov/pubmed/27725916
^[13]=Kobelt, D., Schleef, M., Schmeer, M. et al., Performance of High Quality Minicircle DNA for In Vitro and In Vivo Gene Transfer, Mol Biotechnol (2013) ^[14]= Scott Prahl, Optical Absorption of Hemoglobin, Oregon Medical Laser Center, http://omlc.org/spectra/hemoglobin/ retrieved 2016-10-15
^[15]= Steven L Jacques (2013) Optical properties of biological tissues: a review, Institute of Physics and Engineering in Medicine, http://omlc.org/news/dec14/Jacques_PMB2013/Jacques_PMB2013.pdf
^[16]= Jim Fessenden (2014) Tuning light to kill deep cancer tumors, http://www.umassmed.edu/news/news-archives/2014/10/tuning-light-to-kill-deep-cancer-tumors/ retrieved 2016-10-15
^[17]= Tan, He, Han, Zhou, Optogenetic Immunomodulation: Shedding Light on Antitumor Immunity, Cell Press, TIBTEC 1434