The vectorization

Vectorization principle [1]

The delivery of a therapeutic molecule to an organ, a tissue, or a cell constitutes today a major challenge. From the beginning of the century, the scientist Paul Ehrlich was already dreaming about « magic bullet » able to transport an active principle in a specific way to its action site. Today, Paul Ehrlich’s dream is nearly real thank to pharmaceutical vectorization development.

Several active principles present, indeed, physico-chemical properties (hydrophily, molecular weight etc…) not in favor of the biologic barrier transit. This barrier separates the administration site from the action site. Other active molecules bump into enzymatic barrier which lead to their degradation and their fast metabolism.
In a general way, it is necessary to notice that barriers are very complex systems made of several elements (epithelium, endothelium, cell membrane) and several components (mechanical, physico-chemical and enzymatic barriers). The therapeutic concentration obtained at the action site is only possible to the detriment of an important loss of medicine to other tissues or cells which leads to important toxic effects and sometimes prohibitive.
Moreover, biotechnologies development gives the access to recombinant proteins and cloned genes, in high quantity. In a same way, progress in organic chemistry on support permits oligopeptides and oligonucleotides creation. These molecules are, probably, the base of tomorrow medicines: highly selective in a molecular way, they lead to endogenous metabolites, which mean non-toxic. However, physico-chemical characteristics and biomimetic of these molecules makes them hard to administer. They are, indeed, badly absorbed (at tissue and cell levels) and often quickly degraded and metabolized and unable to reach their target at tissue or cell level. This is one of the principal limits of the development of these molecules as medicine.
For all these reasons, the development of active principle vectors is considerably expanding during last years. Based on new physico-chemical concepts, the galenic research permits to imagine submicronic systems administration, biological or chemical, able to: (i) protect the active molecule from degradation and (ii) control its liberation in time and space.

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Pharmacokinetic modification, pharmacodynamic and bioavability [1,2]

A “classical” medicine is distributed in the organism in function of its physico-chemical properties:

- pKa,
- Lipophily,
- Kd (protein fixation).

Furthermore, a « vectorized » vector is distributed in the organism in function of its vector properties:

- Vector size,
- Vector lipophily,
- Vector global charge,
- Vector stability,
- Vector immunogenicity.

Its distribution, so its bioavability is not the same. The bioavaibility acts directly on active principle efficacy and toxicity. If the injected dose is concentrated in one place, a better efficacy is obtained than if the dose is diluted in the all organism.
Thus, a vectorized medicine is not distributed in the same way and has not the same efficacy and toxicity than a “classical” medicine.
In the case of a “vectorized” medicine, the natural distribution is avoided. It allows to the active principle to be delivered to its target, and to concentrate the dose on the interested surface. Efficacy is increased and the toxicity is decreased.

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Vector components [3,4]

A vector is a nanotransporter composed of several elements.
First, there is a reservoir, which is the active principle storage element. It can be very different in function of the vector nature. For example, it can be composed of phospholipids like in the liposomes’ case, or proteins, as in the viruses’ case. But independently of its nature, the reservoir has a storage function and manages the active principle protection.
Then, there is the targeting system, this system permits to completely change vector bioavability and release preferentially the active principle in the wanted organ, tissue or cell. This system is often made by small proteins or proteins recognized by a ligand membrane exposed to the targeted cell surface.
Finally, the last element is the stealth system. Why, and against who?
A vector, usually bigger than 60nm and potentially charged, is detected by the immune system. It is one of the principal issues of this technology. That is why stealth systems were elaborated.

All vectors do not have the same components, and the same possibilities. Each vector type has its own characteristic.

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Vector types [5,6,7,8,9,10,11,12]

Two big vector classes exist: biological vectors and chemical vectors.

First vector type, viral vector. Viral vectors are viral particles carrying an artificially modified genome, in comparison to viral strain from which is derived the vector.
Viruses represent natural vectors the most evaluated for foreign genetic information transfer in a cell. Many viruses have been adapted in vectors: the most advanced are retroviruses, adenoviruses and adeno-associated viruses (AAV).
Natural viral cycle is divided in two distinct parts: infection and replication. Infection consists in the viral genome introduction into the cell. Viral gene expression leads in a second time to new viral particles formation, this is the replication.
During the production of recombinant virus, viral particles encapsidate a modified genome containing expression cassette of the therapeutic gene of interest at the place of all or a part of the viral genome.
Transduction is defined by an abortive infection by the defective recombinant vector, for the replication, which introduces only functional genetic information in a targeted cell. This vector type has an advantage, it is naturally a genetic information vector, it has then all the capacity to move in the circulation flow, to recognize a targeted marker and to enter the interest cell to integrate the therapeutic gene. But it gets drawbacks as well: its high immunogenicity, the possibility of viral genome recombination in vivo and its industrialization.

Another type of viral vector, which is bacterial: phages. These viruses have others characteristics than eukaryotic vectors. They function on the same way; they are natural vector of genetic information as well. On the other hand, native of the prokaryote world, they are safer to use, easier to produce, and less efficient face to an eukaryotic environment. Their genome should integer specific sequences, which permit them to increase their transfection efficiency. They possess, like other eukaryotic viruses, easiness to integer genetic information of therapeutic interest inside their capsid and an easiness to target a marker. Nevertheless, they enter badly in eukaryotic cells and their immunogenicity is very strong as well.

To finish, last vector of the biological class, bacteria. This vector type, one of the most original, is not very studied and only developed in laboratories. Principally used for protein secretion in vivo, this vector cannot transfect cell of interest. Trials were tested on inflammation secretion factor, or another immunostimulating agent, in tumors. In addition to its inability to transduce genetic information in cell of interest, bacterial vectors are immunogene and potentially dangerous for humans. This vector type still exists because it brings some particular possibilities. The bacterial vector can target a tissue and deliver a therapeutic protein, it is easy to produce as well and in some cases, depending of the strain, it resists to the immune system.

In the second vector class, we find lipidic and polymeric nanoparticle vectors.
Nanoparticle vectors are colloidal systems; their structure is composed of lipids or polymers, biodegradable if possible. Nanoparticles can be matricial; in this case, active principle can be dispersed or dissolved in the matrix and be released only by a simple diffusion or after biodegradation in the organism. Nanoparticles can be a type of reservoir, in this case, they are constituted by a central nucleus usually liquid and surrounded by a thin wall of lipids or polymers which the thickness is not over some nanometers.
Lipidic nanoparticles have the advantage to be biomimetic and biodegrable, moreover, they are not stable, their DNA compaction capacity is 40 to 50 less important than a virus of the same size and they are highly immunogene, even if they have “stealth” molecules (e.g. PEG).
Polymeric nanoparticles are more stable than there lipidic homologous, but their entry in cells is weaker. They have a potential toxicity and are immunogene as well, even if they have “stealth” molecules.

Each vector type has its own properties, but they have some issues as well. Vectorization technologies have several recurrent issues, specific or not to a type of vector.

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Vectorization problems [13,14,15,16,17,18,19]

Today, six major issues still unresolved:

- Stability,
- Toxicity,
- Targeting/specificity,
- Passage through the membrane,
- Immune system: resistance or stealth,
- Industrialization.

The stability is an issue concerning principally lipidic nanoparticles, indeed lipidic autoassembly like liposomes are stable in time. In vivo, this type of vector incorporates or disrupts, and then releases its content. Some molecules, like cholesterol, can improve its stability, but they stay the most fragile vectors and so, the less protector for the active principle.

The toxicity is an issue from two origins. It can result from a potentiality of some vector to become uncontrollable or from fabrication methods of some other vectors. Viruses are probably the best vectors, but they are the most dangerous as well. There is a great potentiality, even for defective viruses, of replication without control in a patient organism, and it is due to the genomic recombination phenomena, very common in this type of biological particles.
The other toxicity issue comes from nanoparticles fabrication methods. The use of organic solvent for their conception can be dangerous for the patient, because even with elimination steps, organic solvent dose injected is never null.

The targeting is considered as a strange issue, it is supposed to be a technological advantage provided by the vectorization, but the targeting is far from being optimal. Targeting is realized by the recognition of a membrane marker expressed at the targeted cell surface. A weak quantity of markers is specific to only one type of cell in the entire organism. So, even if the therapeutic agent dose is higher at targeted cells level, other cells in the body will suffer to the therapeutic agent effects.

The passage of the membrane is a key event for medicine; its efficiency is linked to its capacity to cross this barrier. The therapeutic agent, whose action is subcellular, should enter inside the cell to act. Prokaryotic vectors as well as polymeric nanoparticles are disabled by this crucial step in the medicine becoming.

The immune system is probably the principal issue. It can be both specific and non specific. Indeed, the immune system fixes all what is superior to 60nm and which is in the circulatory flow. It is able to adapt and react faster as one goes along the meeting with vectors. We know for a long time that viruses, phages and bacteria are detected by the immune system and are eliminated. The first detection is always late compared to the vector injection; this permits an efficacy of the first dose. But during the second injection, the clearance is so fast that the vector efficacy is nearly null.
According to some people, this issue is not transposable to nanoparticles, which benefit from stealth molecules. These molecules, like Polyethylen Glycol (PEG) for example, are supposed to avoid recognition by the immune system thanks to a steric repulsion. But this is not true, or at least only in single injection, PEG like other stealth molecules, offers an accessible epitope to antibodies and other body opsonins. Then, during the second injection, an immune response is put in place and clearance is speed up, decreasing considerably the therapeutic efficacy.
However, vectors recognized by opsonins are absorbed by hepatho-splenic system macrophages (liver and spleen), then it is quite easy to target these organs pathologies with this technology. It is totally naturally, via hepatic and splenic macrophages, that organism concentrate “vectorized” medicine dose in these two organs.

Industrialization is an issue from another nature. However, it stays essential to control. Many revolutionary techniques never succeeded due to a disability to be transposed to industrial scale. This can be the case for some type of vectors. Today, except prokaryotic vectors, vector production cost is very high. For example, viral vector has to be produced by eukaryotic cells, which seems to be a brake to their commercialization. For nanoparticles, prices are high for the moment, however some products with hepathosplenic tropism can be found on the market. Moreover, fabrication of more complex vectors, which need more components, several conception steps and organic solvents use has a production cost very high.

To sum up, a table of each vector type faces to the vectorization problems:


We observe in the previous table, that each vector type fails face to at least one of the problematics. So, we can say that this technology, as promising it is considered, is faced to a technological barrier.

This table shows that each vector type possesses at least one problem. But to each problematic, one or more vector reply positively. Viruses, phages and bacteria are biological vectors, so we can associate them to form an optimal vector, replying to all major problematics.

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The Double Vector System

Synthetic biology is based on the principle that all genes, when standardized, can be assembled to form a biological algorithm.
Designed on this principle, the Double Vector System (DVS) is composed of a bacterium, a phage and eukaryotic virus proteins. A complex mix offering, to vectorization technology, a key to its major issues.

DVS components:

DVS2En.png and DVS3En.png

The DVS is composed by a first vector, tissue vector, from bacterial type, able to reach a tissue without being eliminated by the immune system. And by a a second vector, created by the first one, from phage type, able to reach a targeted cell and to integer itself, thanks to viral proteins exposed to its capsid. Once the target is reached, it releases a therapeutic plasmid which has a action against the disease.

DVS mechanism of action:

DVS is not only replying to underlying vectorization problematic, it enhances some intrinsic characteristics of the technology.

Discover in more details each DVS components through pages of the website.

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