FDA cellular & gene therapy guidance

Gene therapy involves the transfer and expression of a therapeutic gene. The nucleic acid transfer needs to have a dominant effect on the cell’s phenotype. Several different mechanisms have been developed to deliver genetic material to living cells. These include various viral and nonviral methods of gene delivery. The viral methods include 1) retrovirus, 2) adenovirus and 3) adeno-associated virus. The non-viral methods of gene delivery include 1) liposomes, 2) particle bombardment, 3) direct injection and 4) electroporation.

Viral vectors has problems associated with safety and limited targeting characteristics. Nonviral physical methods such as electroporation and ultrasound has received much attention. Laser mediated gene transfection is also an attractive physical method for targeted gene therapy because of the high spatial controllability of laser energy.

Intracellular drug delivery has become a rat-limiting stp in many aspects of the pharmaceutical indsutry. This is especially true for delivery of nucleic acids including DNA, mRNA, shRNA, siRNA and gene editing agents, whcih need to access to the cytosol to exert therapeeutic functions. DNA needs to be further translocated into the nucleus for transcription. Nanoparticles are widely investigated vehicles for intracellular drug delivery. To get access into the cytosol, the drugs have to be encapsulated in a carrier, taken up by target cells through processes such as endocytosis or macropinocytosis, and escape from teh endocytic vesicles before being degraded or recylced back outside the cell. (Jiam, Nano Lett, 20(2): 1117-1123,2020).

Physical Delivery Methods:

Physical delivery methods destablize membranes as a mechanisms for introducing genetic material. Importantly, these gene delivery methods need not be used in isolation and there are many examples of combining different methods, especially chemical and physical.

Electrophoration: has been applied extensively in gene transfer, DNA vaccination, and drug delivery, and is the mode of delivery for most plasmids used in recent and ongoing clinical trials.

Major ongoing challenges in electrogene transfer include variable transfection efficiency in different tissues and a lack of targeting. Electroporation also tends to promote high cell toxicity, with extensive cell loss, especially in primary cells, which is limited supply per patient. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

Ultrasound (sonoporation): is another physical approach where pores are generated typically in the presence of microbubbles, exploiting the weak interactions of lipid bilayers to promote acoustic streaming of genetic material into cells. Sonoporation also can facilitate local transport across more complex biological structures, such as skeletal muscle, vasculature, and solid tumors. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

Viral Vectors: (see outline). 

Non-Viral Vectors: (see outline)

Laser Delivery

Laser Mediated Gene Transfer: 

–Laser Delivery of small interfering RNA: Tang (Plant Science 171 (2006) 375-381 disclose efficient delivery of siRNA to plant cells by a nanosecond pulsed laser induced stress wave for postranscriptional gene silencing.

Delivery of RNA:

The functional delviery of RNA is an emerging approach to vaccination and therapy. Although naked mRNA delivery has been reported, its efficacy is limited by high rates of plama RNA degradation, poor cellular entry, and nucleic acid induced inflammatory responses. Formulation of RNAs into lipid nanopartices (LNPs) has been developed as a way to overcome these limitations (see below).

Scientists have developed strategies for increasing mRNA protein yeilds. For example, researchers have been exploring ways to chemically alter mRNAs both within the strand at at their 5′ N7-methylguanosine (m7G) caps in order to further increase stability and efficacy. Protein synthesis relies on an interaction between m7G caps and eukaryotic translation initiation factors (eIFs) like eIF4E. These caps help avoid rNase degradation and promote higher levels of translation. However, sometimes mRNA can become uncapped. Another issue is mRNA half-lives are short; even capped, they can be degraded by ribonucleases (RNases) and other proteins in the cell. This means less time for the cell to convert them into protein. To combat this, researchers have adopted circular mRNA (circRNA) as a popular method of drug delivery. This structure eliminates the ends which make it vulnerable to exonuclease activity. While a lack of RNA ends provides resistance to exonucleases, this also means no cap modifications to promote translation and no easy access for translation macherny to initiate protein synthesis. These RNAs are only translatable via internal ribosome entry sites (IRESs). Thus while circRNA structure is more stable, protein yields are still relatively low. A solution may lie in looking at synthesis methods for other kinds of RNA, like siRNA. The traditional method for mRNA synthesis is compeltey enzymatic, lacking the versatility to control or introduce chemical modifications in a site-specific manner. On the other hand, traditional methods for non-coding RNA synthesis such as siRNA therapeutics are completely chemical, allowing precission.  (Soren Hough, “Playing with LEGO to improve mRNA efficiency” (ct 16, 2024).