Team:UANL Mty-Mexico/project/DNA-Program-Delivery

What is a DNA delivery system?
A DNA delivery system is a method by which exogenous DNA is introduced into an organism. These systems can be divided into two general categories: viral delivery systems and the non viral delivery systems. The viral delivery systems are the ones that use the viral infection mechanism to deliver genetic material, usually in a viral particle incapable of replicating itself. On the other hand, the non viral delivery system comprehends a bigger set of strategies:

• ● Electroporation, which is the introduction of DNA into a cell without a wall using an electric field to form pores in the membrane.
• ● Microinjection is another mechanism which consists in introducing DNA by pressure into an isobaric system.
• ● Lipofection as well is a strategy that introduces genetic material by using particles known as liposomes.
• ● Among others.

DNA Delivery Systems on Eukaryotes

With evidence on induced Pluripotent Stem Cells (iPS cells).

Justification

Cell reprogramming for generation of iPS cells is a technique which uses transduction systems with “reprogrammation factors”, proteins and transcriptions factors involved primarily in the methylation patterns in the genome of a differentiated cell, which are modified in order to produce a cell capable of differentiating again, into different types of cells, but not a whole organism. While this reprogrammation might seem different from the “biohacker” system we propose, it is the same basic principle; the use of a gene/DNA delivery system, in this case, for a mammalian cell; the uses of this biohacker system with this type of DNA delivery system will be discussed, while reprogrammation efficiency cannot be discussed, since it relies on more complex factors, as the complete modification of methylation patterns, the stability of the new methylation patterns and a steady state of other differentiation and growth factors. [12]

Retroviruses.

A common tool, used in both clinical gene therapy and basic research, are retroviruses; their biology is well understood, and they have a high efficiency on both transduction and expression of genes, and can be replication-competent (it has in its genome the essential genes for virion synthesis) and replication defective; thus, the technique is widely used. Nevertheless, the virus genome is large, so it has a limited cloning capacity for multiple genes, which makes a reprogrammation difficult on whole systems or modules on synthetic biology or induction of pluripotent cells. [13]
Even though, in the work of Yamanaka, the first delivery of four reprogrammation factors on rat fibroblasts in order to make iPS was made using a Moloney murine leukemia virus (MMLV) based retrovirus vector, but this system often reactivated the MMLVLTR promoter, causing tumors due to expression of c-Myc factor; when this promoter was removed, they got completely functional iPS cells.
Better results have been reported with other strains of cells, reprogrammation factors or retroviruses.

Lentiviruses.

In contrast with retroviruses, lentiviral systems can infect both dividing and non-dividing cells, whilst retroviral systems can only infect dividing cells. All other features are shared, including retrotranscription and genome integration.
Though lentiviral systems integration is unpredictable, it is less likely that they integrate in oncogenic regions of the genome, causing cancer, than gamma-retroviral systems. Unlike the retroviral systems, all of the lentiviral systems have been designed replication-defective.

Episomal vectors

Most plasmids cannot replicate themselves in a mammalian environment, therefore, they only express transiently. Nevertheless, plasmids like oriP/Epstein-Barr nuclear antigen-1 replicate autonomously as extrachromosomal elements without integration in cells, dividing or non-dividing.

Adenovirus

Adenoviral systems are also non-integrative vectors (except on eggs), can transfect both replicative and non-replicative systems, as well as almost every kind of cell, except for certain lymphoid cells<.

Others

In iPS cells, excisable integrated vectors are also used in order to generate iPS transgene free cells, capable of differentiating, but the biohacker system itself does not consider the excision of the transgenes at any moment, it rather focuses on the introduction of new programs in the same cell, as a way of direct “differentiation”, so excisable integrated vectors were not considered.

Animal-bacteria horizontal gene transfer.

Horizontal gene transfer has been described in a wide variety of organisms, mostly prokaryotes; whole genome analyses show that genes are transferred horizontally between closely related taxa, and between bacteria inhabiting the same environment. [15]
Horizontal gene transfer is also found in eukaryotes, and even on superior animals; examples range from the transfer of P elements between Drosophila melanogaster and D. willistoni, the transfer of genes for carotenoid biosynthesis from fungi to pea aphids and the more recently discovered heritable lateral gene transfer in humans and Trypanosoma cruzi. [16]
Although rare, interkingdom gene transfer has also been observed, the Eubacteria Thermotoga maritime has 81 archeal genes, and the well known Eubacteria-Eukaryota transference; Agrobacterium tumefaciens transfers 10-30 kbp to plants. In the case of bacteria to animals, it happens through symbiosis, like the symbiotic relationship between Wolbachia with arthropods and filarial nematodes.
Though not a DNA delivery system in itself, the danger of transferring the program to other species, bacteria, animals or plants, has to be considered in function of the organism we are using.

Animal yeast transfer systems
Having explained this, there is a delivery system that uses this horizontal gene transfer in an interspecies system, the delivery of DNA and even RNA using recombinant yeast to transduct mammalian phagocytic cells. Even if it was designed to produce DNA vaccines, this system could be used as a DNA delivery system for almost any phagocytic organism, more specifically, protozoan, amoebas being the classic phagocytic example.

The design of a vector capable of replicating in both amoebas and yeasts might seem complicated, but finding a real life application for the idea is even more complex.[11]

Non-living delivery systems.
Justification.
This particular method of DNA delivery is not important for the idea proposed; since the organisms need to be in a controlled environment, in order to be transformed with methods using non-living delivery systems, in particular nanoparticles (e.g. biolistics). Its use as a transformation method on the field is limited. [9][10]

Plant gene transfer systems.
Justification.
The idea proposed in this project is meant to be employed on the field, and since plants are one of the most important GMOs released in the environment, it is necessary to review the delivery systems on plants as well. This, along with bacteria used for bioremediation techniques, would cover the vast majority of GMOs.

Agrobacterium tumefasciens.

Agrobacterium tumefasciens is a species of bacteria widely distributed in soil; it is the oldest and best researched plant transformation method, though we still lack even though we still lack important amounts of knowledge about the basic mechanisms of recognition, attachment, and integration.

Nevertheless, our primary concern about using A. tumefasciens on crops is not its transformation efficiency, but the possibility of disseminating transgenes in the environment. While it is true that A. tumefasciencs is widely distributed in soil, most isolates do not contain this plasmid, and the only genes it can transfer are the vir genes; this genes do not represent an advantage to the host organism, so they do not survive or overcome the rest of the population.

However, if we transform the host organism on the field with an A. tumefasciens circuit, it could also transform other plants, giving them the traits that the circuit could carry, giving them (or not) an advantage over other organisms. But the problem is the capability of the bacteria to transform other organisms.

We cannot change a mechanism that we do not understand, so making the bacterium host-specific is impossible, since we do not know what proteins are involved in host attachment and recognition.[8]

Virus
The use of virus to genetically modify an organism is mostly reduced to promoters and other regulatory elements of the virus, since plant virus are not widely studied (except for TMV); but most of the known ones are host-specific.

In plants, there’s a very special kind of virus, called “virions”, they are RNA particles with no mRNA activity, whose use as a DNA delivery system is null.

Continuing with the virus subject, as in animals, the genetic material of plant virus can be RNA or DNA, never both. Among the RNA virus are the families Tobacovirus (turnip vein-clearing virus, tobacco mosaic virus) and Potexvirus (Alternanthera mosaic virus and potato virus X), and among DNA (which are relevant to us) are the Geminiviridae; small viruses (2.5-3.0 kb per single stranded DNA circle) which replicate within the nucleus, completely depending on host proteins to complete their life cycle.

Which means another transformation method, one involving the modification of certain sequences in the Geminivirus, along with the addition of the relevant vir genes from A. tumefasciens or a transposon linked sequence, and our circuit, can be proposed.This way we would have a host-specific delivery system for plants, which could be transfected by a simple rub-inoculation. The system could be replication-competent or replication-deficient, depending on the modified sequences, and integrative or episomal in function of the number of vir genes added.[7]

Though not a major set-back, the rub-inoculation method has the disadvantage of limiting the number of specimens that can be transformed at one time; a difficulty that the reprogramator method does not have due to its sprayed inoculation approach.[6]

Genetic delivery system using specific virus for plants
A reasonable viral vector option for Maize (Zea mays), one of the most important crops for global agriculture, is the Maize rayadofinomarafivirus (MRFV). These virus, first reported in Zea mays in El Salvador by Ancalmo and Davies (1961), have as its only natural host the Zea mays ssp. mays (maize) and Zea mays ssp. mexicana. It produce chlorotic vein stippling and striping especially in new cultivars, and its transmitted by an insect named Dalbulusmaidis (Cicadellidae). The experimental host range has determined that it have just some few families susceptible, the Gramineae, including three species: Zea mays, Zea mays ssp. mays, and Zea mays ssp. mexicana. Its genome consists of single-stranded RNA with a total genome size of 6.7 kb. Also there are other virus that are good options as gen delivery vectors.

Conclusion
Virus were chosen as our delivery system,since they are the only specific and safe, both integrative and non integrativesystem, which can be released on the field without fearing any cross contamination; making it the only suitable delivery system for the reprogrammation scheme.