New fast and efficient cell-type targeting


It is a remarkable proof for the concept of IOB: working closely hand in hand, our molecular and clinical researchers have developed a library of 230 adeno associated viral vectors (AAVs), each with a different synthetic promoter.

A number of these AAVs specifically target expression to neuronal and glial cell types in the mouse and non-human primate retina in vivo, and in the human retina in vitro.

The library is online, and includes three-dimensional confocal scans.

It can be used to search for cell types labeled by active AAVs.

Different potentials for basic and translational research in mice, NHPs and humans

Targeting genes to specific neuronal or glial cell types is valuable both for understanding and repairing brain circuits.

Credit: Institute of Molecular and Clinical Ophthalmology Basel (IOB)

AAVs are frequently used for gene delivery, not least because they are safe for the use in human gene therapy and allow efficient and long-lasting transgene expression in target cells.

However, targeting expression to specific cell types is an unsolved problem.

“Our resources allow economic, fast and efficient cell-type targeting in a variety of species, both for fundamental science and for gene therapy.

Different neuronal and glial cell types of mice, NHPs and humans can be efficiently targeted with our set of AAVs.

Subsets are particularly useful in basic research for recording or modulating the activities of cell types, others applicable in translational research for gene therapy of cell type specific human diseases such as retinitis pigmentosa and macular degeneration.

We also demonstrate applications for recording and stimulation, as well as for the intersectional and combinatorial labeling of cell types,” says Josephine Jüttner, first author of the study, and head of the viruses platform in the IOB Molecular Research Center.

While the team could not target chosen cell types, it was able to preferentially target outer or inner retinal cell types with a choice of synthetic promoters.

The so-called ProC group of synthetic promoters mostly maintained selectivity across species.

It could be particularly useful for translational applications.

Targeting expression in translational research

Cell-type-targeted modulation of brain function and cell-type-specific gene replacement are repair strategies for treating human diseases.

For example, in advanced retinal cell degeneration in advanced retinal cell degeneration.

Therefore targeting and modulating the remaining retinal circuitry is crucial.

Despite the central importance for both basic and translational research, most current technologies available for cell-type targeting rely on transgenic animals

Either the genetic tool that senses or modulates brain function, or the enzyme which allows the genetic tool to be conditionally expressed, is from the animal’s genome.

This limits their applicability.

Transgenic components in a cell-type targeting strategy exclude its use in therapy for humans, limit its range of application in preclinical, non-human primate (NHP) research and complicate its use in model organisms such as mice.

The development of transgenic NHPs and mice is costly and slow.

Moreover, optimizing cell-type targeting in mice yields vectors that are unlikely to optimally target the same cell type in humans.

The new AAV vector library and approach allows testing cell type targeting in human retinas in vitro.

The IOB molecular and clinical research teams have shown that this significantly increases the probability that the same vector will target the desired cell type in patients in vivo.

Better understanding of vision and the human brain

Culturing postmortem human brain parts, such as the retina or brain slices, in combination with cell-type targeting AAVs, could lead to a better understanding of the organization and function of cell types in circuits in the human brain.

Particularly notable for retinal research is the targeting of photoreceptors with optogenetic tools and other cell types with genetically encoded activity sensors.

Although the natural input from light is lost, restoring light sensitivity to photoreceptors may allow computations within the human retina to be studied for several months at the level of cell types and circuits, making the human retina a simple and translationally relevant model system for research.

The study results suggest that the absence of expression in a given cell type or cell class in mice is a useful proxy for the same in humans; therefore, studies in mice can be used to eliminate AAV vectors to be tested in humans.

The discovery of DNA as the biomolecule of genetic inheritance and disease opened up the prospect of therapies in which mutant, damaged genes could be altered for the improvement of the human condition.

The recent ability to rapidly and affordably perform human genetics on hundreds of thousands of people, and to sequence complete genomes, has resulted in an explosion of nucleic acid sequence information and has allowed us to identify the gene, or genes, that might be driving a particular disease state.

If the mutant gene(s) could be ‘fixed’, or if the expression of overactive/underactive genes could be normalized, the disease could be treated at the molecular level, and, in best case scenarios, potentially be cured.

This concept seems particularly true for the treatment of monogenic diseases, i.e. those diseases caused by mutations in a single gene.

This seemingly simple premise has been the goal of gene therapy for over 40 years.

Until relatively recently, that simple goal was very elusive as technologies to safely deliver nucleic acid cargo inside cells have lagged behind those used to identify disease-associated genes.

One of the earliest approaches investigated was the use of viruses, naturally occurring biological agents that have evolved to do one thing, i.e. deliver their nucleic acid (DNA or RNA) into a host cell for replication.

There are numerous viral agents that could be selected for this purpose, each with some unique attributes that would make them more or less suitable for the task, depending on the desired profile [1].

However, the undesired properties of some viral vectors, including their immunogenic profiles or their propensity to cause cancer have resulted in serious clinical adverse events and, until recently, limited their current use in the clinic to certain applications, for example, vaccines and oncolytic strategies [2].

More artificial delivery technologies, such as nanoparticles, i.e. chemical formulations meant to encapsulate the nucleic acid, protect it from degradation, and get through the cell membrane, have also achieved some levels of preclinical and clinical success.

Not surprisingly, they also have encountered some unwanted safety signals that need to be better understood and controlled [3].

Adeno-associated virus (AAV) is one of the most actively investigated gene therapy vehicles.

It was initially discovered as a contaminant of adenovirus preparations [45], hence its name.

Simply put, AAV is a protein shell surrounding and protecting a small, single-stranded DNA genome of approximately 4.8 kilobases (kb).

AAV belongs to the parvovirus family and is dependent on co-infection with other viruses, mainly adenoviruses, in order to replicate. Initially distinguished serologically, molecular cloning of AAV genes has identified hundreds of unique AAV strains in numerous species.

Its single-stranded genome contains three genes, Rep(Replication), Cap (Capsid), and aap (Assembly).

These three genes give rise to at least nine gene products through the use of three promoters, alternative translation start sites, and differential splicing.

These coding sequences are flanked by inverted terminal repeats (ITRs) that are required for genome replication and packaging.

The Rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40), which are required for viral genome replication and packaging, while Cap expression gives rise to the viral capsid proteins (VP; VP1/VP2/VP3), which form the outer capsid shell that protects the viral genome, as well as being actively involved in cell binding and internalization [6].

It is estimated that the viral coat is comprised of 60 proteins arranged into an icosahedral structure with the capsid proteins in a molar ratio of 1:1:10 (VP1:VP2:VP3) [6].

The aap gene encodes the assembly-activating protein (AAP) in an alternate reading frame overlapping the cap gene.

This nuclear protein is thought to provide a scaffolding function for capsid assembly [7].

While AAP is essential for nucleolar localization of VP proteins and capsid assembly in AAV2, the subnuclear localization of AAP varies among 11 other serotypes recently examined, and is nonessential in AAV4, AAV5, and AAV11 [8].

Although there is much more to the biology of wild-type AAV, much of which is not fully understood, this is not the form that is used to generate gene therapeutics.

Recombinant AAV (rAAV), which lacks viral DNA, is essentially a protein-based nanoparticle engineered to traverse the cell membrane, where it can ultimately traffic and deliver its DNA cargo into the nucleus of a cell.

In the absence of Rep proteins, ITR-flanked transgenes encoded within rAAV can form circular concatemers that persist as episomes in the nucleus of transduced cells [9]. Because recombinant episomal DNA does not integrate into host genomes, it will eventually be diluted over time as the cell undergoes repeated rounds of replication.

This will eventually result in the loss of the transgene and transgene expression, with the rate of transgene loss dependent on the turnover rate of the transduced cell.

These characteristics make rAAV ideal for certain gene therapy applications. Following is an overview of the practical considerations for the use of rAAV as a gene therapy agent, based on our current understanding of viral biology and the state of the platform.

The final section provides an overview for how rAAV has been incorporated into clinical-stage gene therapy candidates, as well as the lessons learned from those studies that can be applied to future therapeutic opportunities.Go to:

Adeno-Associated Virus (AAV) Vector Designs

The main point of consideration in the rational design of an rAAV vector is the packaging size of the expression cassette that will be placed between the two ITRs. As a starting point, it is generally accepted that anything under 5 kb (including the viral ITRs) is sufficient [10].

Attempts at generating rAAV vectors exceeding packaging cassettes in excess of 5 kb results in a considerable reduction in viral production yields or transgene recombination (truncations) [11].

As a result, large coding sequences, such as full-length dystrophin, will not be effectively packaged in AAV vectors.

Therefore, the use of dual, overlapping vector strategies (reviewed by Chamberlain et al.) [12], should be considered in these cases. An additional consideration relates to the biology of the single-stranded AAV-delivered transgenes.

After delivery to the nucleus, the single-stranded transgene needs to be converted into a double-stranded transgene, which is considered a limiting step in the onset of transgene expression [13].

An alternative is to use self-complementary AAV, in which the single-stranded packaged genome complements itself to form a double-stranded genome in the nucleus, thereby bypassing that process [1314]. Although the onset of expression is more rapid, the packaging capacity of the vector will be reduced to approximately 3.3 kb [1314].

AAV2 was one of the first AAV serotypes identified and characterized, including the sequence of its genome. As a result of the detailed understanding of AAV2 biology from this early work, most rAAV vectors generated today utilize the AAV2 ITRs in their vector designs.

The sequences placed between the ITRs will typically include a mammalian promoter, gene of interest, and a terminator (Fig. 1).

In many cases, strong, constitutively active promoters are desired for high-level expression of the gene of interest. Commonly used promoters of this type include the CMV (cytomegalovirus) promoter/enhancer, EF1a (elongation factor 1a), SV40 (simian virus 40), chicken β-actin and CAG (CMV, chicken β-actin, rabbit β-globin) [15]. All of these promoters provide constitutively active, high-level gene expression in most cell types. Some of these promoters are subject to silencing in certain cell types, therefore this consideration needs to be evaluated for each application [16].

For example, the CMV promoter has been shown to be silenced in the central nervous system (CNS) [16].

It has been observed that the chicken β-actin and CAG promoters are the strongest of these constitutive promoters in most cell types; however, the CAG promoter is significantly larger than the others (1.7 kb vs. 800 bp for CMV), a consideration to take into account when packaging larger gene inserts [15].

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Fig. 1
Schematic representation of the basic components of a gene insert packaged inside recombinant AAV gene transfer vector. AAV adeno-associated virus, ITR inverted terminal repeat

Although many therapeutic strategies involve systemic delivery, it is often desirable to have cell- or tissue-specific expression.

Likewise, for local delivery strategies, undesired systemic leakage of the AAV particle can result in transduction and expression of the gene of interest in unwanted cells or tissues.

The muscle creatine kinase and desmin promoters have been used to achieve high levels of expression, specifically in skeletal muscle, whereas the α-myosin heavy chain promoter can significantly restrict expression to cardiac muscle [1517].

Likewise, the neuron-specific enolase promoter can attain high levels of neuron-specific expression [1819]. Often is the case, systemic delivery of AAV results in a significant accumulation in the liver. While this may be desirable for some applications, AAV can also efficiently transduce other cells and tissues types.

Thus, in order to restrict expression to only the liver, a common approach is to use the α1-antitrypsin promoter [2021]. Finally, there are now technologies that have the ability to generate novel, tissue-specific promoters, based on DNA regulatory element libraries [22].

Over the course of the past 10–15 years, much work has been done to understand the correlation between codon usage and protein expression levels.

Although bacterial expression systems seem to be most affected by codon choice, there are now many examples of the effects of codon engineering on mammalian expression [23].

Many groups have developed their own codon optimization strategies, and there are many free services that can similarly provide support for codon choice.

Codon usage has also been shown to contribute to tissue-specific expression, and play a role in the innate immune response to foreign DNA [2425]. With regard to the gene of interest, codon engineering to support maximal, tissue-specific expression should be performed.

Additionally, terminator/polyadenylation signal choices, the inclusion of post-transcriptional regulator elements and messenger RNA (mRNA) stability elements, and the presence of microRNA (miRNA) target sequence in the gene cassette can all have effects on gene expression [26].

The human factor IX 3′ UTR, for example, was shown to dramatically increase factor IX expression in vivo, especially in the context of additional cis regulatory elements [27]. Likewise, synthetic miRNA target sequences have been engineered into the 3′ UTR of AAV-delivered genes to make them susceptible to miRNA-122-driven suppression in the liver [28].

Although there is much known about these individual components that needs to be considered when designing an AAV vector, the final design will most likely need to be determined empirically.

It is not yet possible to know how a particular design will function by just combining the best elements together based on published reports, therefore considerable trial and error will eventually be required for deciding on the final construct.

In addition, one also needs to consider the differences between in vitro and in vivo activity. Although it is possible to model rAAV expression in rodents, there is still significant concern about the translatability to humans.

More information: Josephine Jüttner et al. Targeting neuronal and glial cell types with synthetic promoter AAVs in mice, non-human primates and humans, Nature Neuroscience (2019). DOI: 10.1038/s41593-019-0431-2

Journal information: Nature Neuroscience
Provided by Institute of Molecular and Clinical Ophthalmology Basel (IOB)


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