2022 has already been a record year for the approval of new gene therapies to treat rare diseases and cancers. More than 2,000 trials are ongoing at the end of June 2022, a slight decrease from the end of 2021. North America leads with about 40%* of active clinical trials, and Europe ranks third with 16%*.

Cell-based immuno-oncology (CAR T cells) represents 34%* of active clinical trials and gene therapies 18%*.

Lentiviral vectors are largely used for ex vivo cell therapies

Lentiviral and retroviral vectors are the most common vector family accross ex vivo gene therapy and immuno-oncology. Adeno associated virus (AAV) ranks second but the first for in vivo gene therapy trials.

Ex vivo use of lentiviral vectors in immuno-oncology is consisting in engineering of T cells with chimeric antigen receptors (CARs) recognizing surface antigens on tumor cells. This use has demonstrated its effective therapeutic benefit for patients with B cell hematological malignancies. Based on this fantastic proof of concept, the focus is now on CAR activity improvement, expanding the approach to solid tumors and simplifying the manufacturing process/clinical protocol. One future avenue is the direct in vivo transfer of the CAR coding sequence into specific immune cells, allowing direct CAR gene delivery. Ex vivo generated CAR T cells have proven their efficacy, we now need a proof of effectiveness with such cells generated in vivo.

 

Lentiviral vectors are now evolving to in vivo therapies

Following the recent SARS CoV2 pandemy emerged end of 2019, numerous COVID-19 vaccines using mRNA, recombinant proteins, replicating or non-replicating viral vector have been quickly developped for fighting the Coronavirus. Ku et al. (2021) have used a powerful in vivo vaccination strategy based on the ability of lentiviral vector to transduce proliferating and non-dividing cells such as dentritic cells. A previous study using a single injection of lentiviral vector expressing the Zika envelope protein protected quickly and durably against Zika infection (Ku et al. 2020). Based on the non-inflammatory, effective and non immunogenic properties of the lentiviral vector, Ku et al. (2021) demonstrated that lentivirus are safe and favorable in vivo delivery methods for mucosal immuzation, which is particularly wanted for immune protection against respiratory infectious diseases like SARS CoV2. Mucosal immunization with lentiviral vectors in boost allows a sterilizing protection, indicating that prime systemic vaccination with lentiviral vector displays a high prophylactic efficacy.

In vivo use of lentiviral vectors (LVs) in immuno-oncology is expected to expand the use of this highly efficient delivery technology into clinic. Today only two clinical programs have been conducted with lentivirus for Parkinson disease in 2004 and DMLA in 2019 with no adverse events related to the delivery tool LV (VSV-G pseudotyped). It will be facilitated by the fact that the lentiviral platform is already validated into clinic as a Starting Material but process must be implemented to reach the Drug Product specifications.

In an original approach, Sobecki M, et al. (2022) successfully used lentiviral vectors as T-cell vaccines for direct in vivo expression of two genes linked to fibrosis development in liver and lungs in mice, paving the way for such lentiviral vectors use for human therapy.

 

Advantages and challenges of lentiviral vector versus AAV

Currently, Adeno Associated Vectors are preferred for in vivo use, despite several drawbacks. Indeed, several AAV-based approaches have been successfully used in adult patients with hemophilia and other diseases but the efficiency of such therapy in humans is hampered by natural exposure to the wild-type virus which leads to the generation and maintenance of neutralizing antibodies to the viral vector in patients. In the human setting, Lentiviral vectors hold several potential benefits :

• First, chances of an immune response curtailing the therapy's efficiency are low thanks to the low prevalence of HIV infection in humans.
• Second, lentiviral vectors are better suited for the treatment of certain diseases due to their ability to carry larger gene inserts.

Moreover, increasing studies over the past two decades have shown that lentiviral vectors modified with different glycoproteins from a donating virus, a process referred to as pseudotyping, have altered tropism and display cell-type specificity in transduction, leading to selective tumor cell killing (Deng et al. 2022).

 

Pseudotyping of lentiviral vectors

VSV-G is not the only one viral glycoprotein ; many others have been incorporated into lentivirus to improve their infectivity and even confer them a more selective tropism, like the gibbon ape leukemia virus (GALV) or the cat endogenous retrovirus envelopes (RD114) (Gutierrez-Guerrero et al. 2020).

Alternative envelopes to classical VSV-G

Others envelope proteins have been used to retarget various cell types such as H and F proteins from the measles virus (MV). More recently glycoproteins derived from other vesiculovirus subfamilies, Cocal, Piry and Chandipura viruses or the VSV New Jersey strain as well as the Nipah virus and other rhabdoviruses proteins have been considered as alternative envelopes for lentivirus pseudotyping. Munis et al. (2018) suggested that hey could have an advantage over the commonly used VSV-G Indiana strain. However, it is difficult to obtain high-titer vectors with some of these glycoproteins, though they transduce efficiently specific cell lineages like hematopoietic cells. 

New innovating way to bring specificity

Finally, to improve specificity of lentiviral vector the pseudotype can be engineered at the surface of the particles to obtain a specific cell targeting. Kasaraneni et al. (2018) have also shown that another way to improve the lentiviral vector tropism to target hematopoietic cells is the incorporation of cell targeting proteins (CTPs) into the viral envelope's outer domain. For this purpose, CTP molecules can be single-chain variable fragments (scFvs). Hence, ScFvs and DARPins have been introduced successfully in MV (Friedrich et al. 2013) and in Nipah virus envelope glycoproteins (Morizono et al. 2005 ; Bender et al. 2016 ; Agarwal et al. 2019) to target oncolytic domains and hematopoietic cells in vitro and in vivo (Gutierrez-Guerrero et al. 2020).

 

A new era for lentiviral vectors as in vivo therapies

The increasing number of clinical trials utilizing CAR-T cell approaches and their already successful application in cancer patients makes this class of biotherapeutics likely to dominate the Cell and Gene Therapy market for the next few years.

In vivo genetic delivery mediated by a lentiviral platform offers novel therapeutics approaches leading to cutting-edge medicines not only for cancer, but also for regenerative medicine and vaccination. Such platform allows both DNA or RNA delivery and can now also be implemented to target specific cells.

 

 

Florine Samain  Cell engineering Project Manager Flash Therapeutics expert since 2016

 

References :

* Source : www.alliancerm.org

Sobecki M., et al. Vaccination-based immunotherapy to target profibrotic cells in liver and lung, Cell Stem Cell, (2022), ISSN 1934-5909, https://doi.org/10.1016/j.stem.2022.08.012.

Agarwal S, et al. In vivo generated human CAR T cells eradicate tumor cells. Oncoimmunology. 2019 Oct 10;8(12):e1671761. doi: 10.1080/2162402X.2019.1671761. PMID: 31741773; PMCID: PMC6844313.

Bender RR, et al. Receptor-Targeted Nipah Virus Glycoproteins Improve Cell-Type Selective Gene Delivery and Reveal a Preference for Membrane-Proximal Cell Attachment. PLoS Pathog. 2016 Jun 9;12(6):e1005641. doi: 10.1371/journal.ppat.1005641. PMID: 27281338; PMCID: PMC4900575.

Friedrich K, et al. DARPin-targeting of measles virus: unique bispecificity, effective oncolysis, and enhanced safety. Mol Ther. 2013 Apr;21(4):849-59. doi: 10.1038/mt.2013.16. Epub 2013 Feb 5. PMID: 23380817; PMCID: PMC3616535.

Gutierrez-Guerrero A, Cosset FL, Verhoeyen E. Lentiviral Vector Pseudotypes: Precious Tools to Improve Gene Modification of Hematopoietic Cells for Research and Gene Therapy. Viruses. 2020 Sep 11;12(9):1016. doi: 10.3390/v12091016. PMID: 32933033; PMCID: PMC7551254.

Kasaraneni, N., et al. A simple strategy for retargeting lentiviral vectors to desired cell types via a disulfide-bond-forming protein-peptide pair. Sci Rep 8, 10990 (2018). https://doi.org/10.1038/s41598-018-29253-5

Ku MW, et al. Intranasal vaccination with a lentiviral vector protects against SARS-CoV-2 in preclinical animal models. Cell Host Microbe. 2021 Feb 10;29(2):236-249.e6. doi: 10.1016/j.chom.2020.12.010. Epub 2020 Dec 16. PMID: 33357418; PMCID: PMC7738935.

Longfei D. et al. Pseudotyped lentiviral vectors: Ready for translation into targeted cancer gene therapy? Genes & Diseases, 2022 https://doi.org/10.1016/j.gendis.2022.03.007.